Macrocyclic metal complexes and their use for the production of conjugates with biomolecules

ABSTRACT

The invention relates to macrocyclic metal complexes and their production and use for the production of conjugates with biomolecules. The conjugates are suitable as contrast media in NMR diagnosis and radiodiagnosis as well as for radiotherapy. High relaxivity is achieved by a special liganding of macrocyclic compounds, and a fine-tuning of the relaxivity is made possible.

The invention relates to the subjects that are characterized in theclaims, i.e., macrocyclic metal complexes as well as their use for theproduction of conjugates with biomolecules. The conjugates are suitablefor the production of contrast media for NMR diagnosis andradiodiagnosis as well as agents for radiotherapy.

A prerequisite for a specific and successful therapy is an exactdiagnosis. Specifically in the diagnostic field, the possibilities havevery greatly increased in recent years, whereby, for example, NMRdiagnosis is able to visualize virtually any anatomical detailselectively and with great accuracy. In many cases, the correspondingstructures are visible only by the application of contrast media,however. Moreover, the possibility exists of configuring the contrastmedia in such a way that they selectively accumulate in the desiredtarget structures. To this end, the accuracy of the imaging can beincreased with simultaneous reduction of the required amount of contrastmedium.

As contrast media for NMR diagnosis, chelate complexes of paramagneticmetals are suitable. The theory and application of gadolinium(III)chelates as NMR contrast media are explained in detail in a surveyarticle by P. Caravan et al. in Chem. Rev. 1999, 99, 2293-2352.

The image intensity in the proton NMR is basically determined by thewater protons. It depends on the nuclear relaxation times. Complexes ofparamagnetic transition metals and lanthanoids shorten the relaxationtimes of adjacent protons by dipolar interactions. The paramagneticcontrast media are not directly detected, but rather an indirectdetection is carried out based on the fact that the contrast media canchange relaxation times of adjacent protons, such as water protons.Based on their high magnetic moments and relaxation efficiency, Gd³⁺,Fe³⁺ and Mn²⁺ are preferred paramagnetic metal cations in NMR diagnosis.

An important physical value, which describes the relaxation behavior ofprotons, is longitudinal relaxation time T₁. Tissues with shortrelaxation times T₁ generally yield images of higher intensity thanthose with longer relaxation times. If the reciprocal value of measuredrelaxation time T₁ based on concentration c is applied to a specificparamagnetic ion, straight lines of rise R are obtained. This rise isalso named relaxivity, which is a measurement of the capacity of thecorresponding paramagnetic ion to shorten the relaxation time of theadjacent protons.

The use of radiopharmaceutical agents for diagnostic and therapeuticpurposes has also been known for a long time in the area of biologicaland medical research. In particular, radiopharmaceutical agents are usedto visualize specific structures such as, for example, the skeleton,organs or tissues. The diagnostic application requires the use of suchradioactive agents, which accumulate after administration specificallyin the structures in patients that are to be examined. These locallyaccumulating radioactive agents can then be traced, plotted orscintigraphed using suitable detectors, such as, for examplescintillation cameras or other suitable recording processes. Thedispersion and relative intensity of the detected radioactive agentidentifies the site of a structure in which the radioactive agent isfound and can visualize the presence of anomalies in structures andfunctions, pathological changes, etc.

Radiopharmaceutical agents can be used in a similar way as therapeuticagents to irradiate pathological tissues or areas. Such treatmentrequires the production of radioactive therapeutic agents thataccumulate in certain structures, organs or tissues.

Because of their sometimes relatively high toxicity, the required ionsare normally not administered in the form of water-soluble salts, butrather in the form of chelate complexes. The latter can be eliminatedvirtually unchanged from the body. The smaller the complexes in solutionare, the lower is their moment of inertia and the faster they rotate insolution (Tumbling Motion Time). The faster a complex rotates, the lowerits relaxivity is. The relaxivity thus increases with the molecular massof the entire complex. A high molecular mass can be achieved by bindingto macromolecules. A good NMR contrast medium is distinguished, i.a., inthat it has a large value for the relaxivity.

Conjugates of Gd-DTPA (diethylenetriaminepentaacetic acid) with albuminare described by, for example, M. D. Organ et al. in Invest. Radiol.1987, 22, 665-671 and U. Schmiedl et al. in Radiology 1987, 162,205-210. Conjugates of macrocyclic metal complexes and biomolecules aredisclosed in WO 95/31444. To improve the selectivity of contrast media,WO 01/08712 proposes a contrast medium that comprises at least two metalchelate units as image-improving groups and at least two “target bindingunits” for binding the contrast medium molecule to the desired targetmolecule or target organ in the body.

Large contrast medium molecules with high molar mass are obtainedaccording to WO 97/02051 by incorporation of macrocyclic metal complexesin cascade polymers.

Tetraazacyclododecanetetraacetic acid derivatives of high stability andgood solubility based on deficient charge that are suitable for bindingto biomolecules are described in EP-A-0 565 930.

The binding of macrocyclic metal complexes to biomolecules that isdescribed above makes possible both an increase of relaxivity andselectivity of the contrast medium. The higher the relaxivity of thecontrast medium, the smaller amount of contrast medium must beadministered to the patient and the greater the opacification in theimage. For this reason, it is additionally desirable to make availableNMR contrast media with the highest possible relaxivity.

An object of this invention thus consists in making available improvedcontrast media for NMR diagnosis and radiodiagnosis as well as agentsfor radiotherapy. In particular, these NMR contrast media are to have ashigh a relaxivity as possible and are to accumulate as selectively aspossible at a desired site in the body.

It has now been found that this object can be achieved, surprisinglyenough, in that a 1,4,7,10-tetraazacyclododecane macrocyclic compoundwith special ligands is provided. The new qualities of the compoundsaccording to the invention are evident if they are bonded tobiomolecules. By the special liganding of the macrocyclic compound, therelaxivity of the contrast medium that is obtained is increased, and inaddition a fine-tuning of the relaxivity for a desired use is possible.

This invention thus relates to compounds of formula I

in which

-   -   Z represents a hydrogen atom or at least two Z's represent a        metal ion equivalent,    -   B¹, B², B³, B⁴ are independently selected from the group        consisting of hydrogen atoms and C₁₋₄-alkyl radicals,    -   R¹, R², R³ are independently selected from the group consisting        of hydrogen atoms and straight, branched or cyclic, saturated or        unsaturated C₁₋₁₀-alkyl or aryl radicals, which optionally are        substituted with a carboxyl group —SO₃H or —PO₃H₂, and whereby        the alkyl chains of the C₁₋₁₀-alkyl radicals optionally contain        an aryl group and/or 1-2 oxygen atoms, provided that at least        one of the radicals B¹, B², B³, B⁴, R¹, R² and R³ does not        represent a hydrogen atom,    -   A represents a straight or branched, saturated or unsaturated        C₁₋₃₀-hydrocarbon chain that optionally contains 1-5 oxygen        atoms, 1-5 nitrogen atoms and/or 1-5 —NR′ radicals, in which R′        is defined as R¹, R² and R³ but can be selected independently,        which optionally is substituted with 1-3 carboxyl groups, 1-3        —SO₃H, 1-3 —PO₃H₂ and/or 1-3 halogen atoms, in which optionally        1-3 carbon atoms are present as carbonyl groups, whereby the        chain or a portion of the chain can be arranged concentrically,        and which is configured in such a way that X is connected via at        least 3 atoms to the nitrogen to which A is bonded, and    -   X represents a group that can participate in a reaction with a        biomolecule, as well as their salts and their use for the        production of a conjugate with a biomolecule.

A corresponding macrocyclic compound in which the four nitrogen atoms ofthe macrocyclic ring are substituted in each case with the substituent—CH(CO₂H)CH₂CH₂—CO₂H is disclosed in P. Caravan et al., Chem. Rev. 1999,99, 2293-2352. A possible use of this compound for the production ofconjugates with biomolecules is not disclosed, however. WO 97/02051discloses macrocyclic compounds, in which A is a radical—CH(R⁷)—CO—NR⁵—U⁶, as intermediate compounds for the production ofcascade polymers. EP-A-0-565 930 discloses macrocyclic compounds, inwhich A is a radical —CH(R⁶)—C(O)—NH—(CH₂)₁₋₆—NH-D-. An increase inrelaxivity by certain substituents is not disclosed. These compounds areconsequently excluded in the definition of the compound of formula I inclaim 1.

Unless otherwise indicated, “alkyl radical” is defined here as asaturated or unsaturated, straight-chain or branched or cyclic alkylradical with the indicated number of carbon atoms. If this radical cancontain other groups or atoms, it is understood here that the othergroups or atoms in addition to the already existing atoms of the radicalare present and can be introduced at any position of the radicalincluding the terminal positions.

“Aryl” is defined here preferably as phenyl, bisphenyl, pyridyl,furanyl, pyrrolyl and imidazolyl. Especially preferred is phenyl.

“Hydrocarbon chain,” which can be arranged completely or partiallyconcentrically, is defined here preferably as a hydrocarbon chain suchas, for example, an alkyl chain, which can comprise, for example, analiphatic or aromatic, optionally heterocyclic 5- or 6-ring (e.g.,phenyl(ene), pyridyl(ene) or cyclohexyl(ene)) or consists of the latter.

In the compound of formula I according to the invention, three of thefour nitrogen atoms of the macrocyclic ring are substituted withoptionally substituted acetic acid or carboxylate methyl radicals. Theseradicals contribute to the coordination or to the charge equalization ofa coordinated metal ion. Z therefore stands either for a hydrogen atomor a metal ion equivalent.

The acetic acid or carboxylate methyl radicals at three of the nitrogenatoms of the macrocyclic ring in addition can have substituents R¹, R²and R³. Moreover, the macrocyclic ring can have substituents B¹, B², B³and B⁴ at four of its carbon atoms. A special feature of the compoundsaccording to the invention consists in that at least one of the B¹, B²,B³, B⁴, R¹, R² and R³ does not represent a hydrogen atom, i.e., themacrocyclic ring must have additional substituents either directly onits ring atoms and/or on the acetic acid or carboxylate methylsubstituents of its nitrogen atoms. By the suitable selection of theseadditional substituents, the desired fine-tuning of the relaxivity of acontrast medium that is produced with use of the compound according tothe invention is carried out.

B¹, B², B³ and B⁴ can be hydrogen atoms or C₁₋₄-alkyl radicals.Preferred C₁₋₄-alkyl radicals are methyl, ethyl and iso-propyl.

If B¹, B², B³ and B⁴ are hydrogen atoms in the compounds of formula Iaccording to the invention, R¹, R² and R³ are independently selectedfrom the group consisting of hydrogen atoms, straight, branched and/orcyclic, saturated or unsaturated C₁₋₁₀-alkyl (preferably C₅₋₁₀-alkyl) oraryl radicals, which optionally are substituted with a carboxyl group,—SO₃H or —PO₃H₂, and whereby the alkyl chains of the C₁₋₁₀-alkylradicals optionally contain an aryl group and/or 1-2 oxygen atoms,provided that at least one of the radicals R¹, R² and R³ does notrepresent a hydrogen atom. As alkyl radicals, straight-chain orbranched, preferably saturated C₁₋₁₀- and especially C₁₋₄-alkylradicals, such as methyl, ethyl, n-propyl, iso-propyl, n-butyl,iso-butyl and tert-butyl, as well as cyclohexyl, are preferred. As analternative, straight-chain, branched or cyclic, preferably saturatedC₅₋₁₀-alkyl radicals, such as pentyl, hexyl, cyclohexyl, heptyl, octyl,nonyl and decyl, are preferred. The C₁₋₁₀-alkyl radicals for R¹, R² andR³ can optionally be substituted with a carboxyl group, —SO₃H or —PO₃H₂.Preferred examples of such substituted alkyl groups are —CH₂—COOH and—C(CH₃)₂—COOH. Moreover, the alkyl chain of the C₁₋₁₀-alkyl radicals cancontain an aryl group and/or 1-2 oxygen atoms. The aryl group and theoxygen atoms can be present at any position within the alkyl chain. Thearyl group, moreover, can also be arranged in terminal position on thealkyl chain and can form an aryloxy group together with an oxygen atom.Especially a phenyl group is suitable as an aryl group.

Preferred alkyl chains for R¹, R² and R³, which optionally contain anaryl group and 1-2 oxygen atoms, are radicals of formula—(CH₂)_(m)—(O)_(n)-(phenylene)_(p)-Y, in which m is an integer from 1-5,n is 0 or 1, p is 0 or 1 and Y is a hydrogen atom, a methoxy radical, acarboxyl group, —SO₃H or —PO₃H₂. Substituent Y is preferably inpara-position in this case.

The aryl radicals for R¹, R² and R³ are preferably phenyl radicals,which are optionally substituted with a carboxyl group, —SO₃H or —PO₃H₂.

If B¹, B², B³ and B⁴ are hydrogen atoms, R¹, R² and R³ are preferablyindependently selected from the group consisting of hydrogen atoms,isopropyl, isobutyl, tert-butyl, a straight-chain or branchedC₅₋₁₀-alkyl radicals, cyclohexyl, —CH₂—COOH, —C(CH₃)₂—COOH, phenylradicals or radicals of formula —(CH₂)_(m)—(O)_(n)-(phenylene)_(p)-Y, inwhich m is an integer from 1 to 5, n is 0 or 1, p is 0 or 1, and Yrepresents a hydrogen atom, a methoxy radical, a carboxyl group, —SO₃Hor —PO₃H₂, and R¹, R² and R³ are especially preferably independentlyselected from the group consisting of hydrogen atoms, isopropyl,cyclohexyl or phenyl radicals, provided that at least one of theradicals R¹, R² and R³ does not represent a hydrogen atom.

The substituted macrocyclic ring of the compound of formula I can bebonded via a spacer A to a biomolecule using a group X, which canparticipate in a reaction with a biomolecule.

In this case, spacer A represents a straight or branched, saturated orunsaturated C₁₋₃₀ hydrocarbon chain, which optionally contains 1-5oxygen atoms, 1-5 nitrogen atoms and/or 1-5 —NR′ radicals, in which R′is defined as R¹, R² and R³ above but can be selected independently,which optionally is substituted with 1-3 carboxyl groups, 1-3 —SO₃H, 1-3—PO₃H₂ and/or 1-3 halogen atoms, in which optionally 1-3 carbon atomsare present as carbonyl groups, whereby the chain or a portion of thechain can be arranged concentrically and which is configured in such away that X is connected via at least 3 atoms to the nitrogen atom towhich A is bonded.

The spacer is to have at least three atoms and preferably at least fouratoms in a chain between the nitrogen atom of the macrocyclic ring andX. A chain of atoms is defined in this case as the shortest connectionbetween the nitrogen atom of the macrocyclic ring and X via a ring aswell. In terms of this definition, for example, a para-phenylene groupwould be regarded as a spacer with four atoms in a chain, and ameta-phenylene group would be regarded as a spacer with three atoms in achain. In determining the length of the atom chain, carbon, nitrogen andoxygen atoms are simultaneously counted in each case as an atom.Substituents in these atoms or side chains are not part of the number ofatoms inside the chain.

-A-X is preferably selected to be different from the substituents—CH(R¹)—CO₂Z, —CH(R²)—CO₂Z and —CH(R³)—CO₂Z.

Spacer A preferably can be represented as a radical A′-U, in which A′ isbonded to the nitrogen atom of the macrocyclic ring and U is bonded toX. Hereinafter, A′ is preferably

-   -   a) a bond,    -   b) —CH(CO₂H)—,    -   c) a group of formula    -   in which Q represents a hydrogen atom, a C₁₋₁₀-alkyl radical,        which optionally is substituted with a carboxyl group, or Q        represents an aryl radical, which optionally is substituted with        a carboxyl group, a C₁₋₁₅-alkoxy group, an aryloxy group or a        halogen atom, and R′ is defined as R¹, R² and R³, but can be        selected independently, or    -   d) a group of formula    -   in which o is 0 or 1, and the ring optionally is annellated with        a benzene ring, whereby this benzene ring, if present, can be        substituted with a methoxy or carboxyl group, —SO₃H or —PO₃H₂.        In the groups above under c) and d), the positions that are        marked    -    are bonded to the adjacent groups, position α is bonded to a        nitrogen atom of the macrocyclic ring, and position β is bonded        to U.

In the group of formula

Q is preferably a linear or branched C₁₋₁₀ radical, especially aC₁₋₄-alkyl radical, such as methyl, ethyl or isopropyl, or a cyclohexylradical. These radicals can optionally be substituted with a carboxylgroup, whereby a carboxymethyl radical is preferred. The preferred arylradical for Q is phenyl. This aryl radical can be substituted with acarboxyl group, a C₁₋₁₅-alkoxy group, an aryloxy group, such asespecially a phenoxy group, or a halogen atom, such as fluorine,chlorine, bromine or iodine, and especially fluorine or chlorine. If thearyl radical is a phenyl radical, the latter is preferably substitutedin para-position with one of the above-mentioned groups. Especiallypreferred groups for Q are methyl, phenyl and p-dodecanoxyphenyl.

R′is defined as R¹, R² and R³ above, but can be selected independentlyfrom R¹, R² and R³. R′ is especially preferably a hydrogen atom.

A′ is preferably selected from a bond, —CH(CO₂H)—, —C(CH₃)H—CO—NH—,—C(phenyl)H—CO—NH—, —C(p-dodecanoxyphenyl)H—CO—NH—,

in which R⁴ is —OCH₃, —CO₂H, —SO₃H or —PO₃H₂.

If spacer A is represented as a radical A′-U, and A′ has the meaningdefined above, U is preferably a straight or branched, saturated orunsaturated C₁₋₃₀-hydrocarbon chain, which optionally contains 1-3oxygen atoms, 1-3 nitrogen atoms and/or 1-3 —NR″ radicals, in which R″is defined as R¹, R² and R³ above, but can be selected independently,and in which optionally 1-3 carbon atoms are present as carbonyl groups,whereby the chain or a portion of the chain can be arrangedconcentrically. U is especially preferably an aryl radical or aC₁₋₂₀-alkyl radical (preferably straight-lined or at least partiallycyclic and saturated) that optionally contains 1-3 oxygen atoms, 1-3 NR″radicals, 1-2 phenylene radicals and/or a pyridylene radical, in whichoptionally 1-3 carbon atoms are present as carbonyl groups, and whichoptionally is substituted with an aryl radical (e.g., phenyl). A′ and Utogether must be configured in such a way that X is connected by atleast three atoms to the nitrogen atom to which A′ is bonded. The chainof at least three atoms is defined as above in A.

The aryl radical for U is preferably a phenyl radical. The C₁₋₂₀-alkylradical for U is preferably a linear, saturated C₁₋₁₀-alkyl radical,cyclohexyl radical or cyclohexyl-C₁₋₅-alkyl radical. The alkyl radicalsof these radicals can optionally be interrupted by 1 oxygen atom, 1phenylene radical and/or 1 pyridylene radical or can contain a —CO—NR″radical or can be substituted with phenyl. U is preferably selected from—CH₂—, —(CH₂)₅—, —(CH₂)₁₀—, -phenylene-O—CH₂—, -phenylene-O—(CH₂)₃—,-phenylene-O—(CH₂)₁₀—, —CH₂-phenylene-, -cyclohexylene-O—CH₂—,-phenylene-, —C(phenyl)H—, —CH₂-pyridylene-O—CH₂—, —CH₂-pyridylene- and—CH₂—CO—NH—CH₂—CH₂—. In the above-mentioned preferred groups for U, thephenylene groups are preferably substituted in para-position, and thepyridylene groups are preferably pyrid-2,5-ylene groups orpyrid-2,4-ylene groups.

Preferred groups for the spacer A are:

Via spacer A, a group X is bonded to the macrocyclic ring in thecompounds of formula I. This group X is a group that can participate ina reaction with a biomolecule. For this purpose, for example, carboxyl(—COOH), activated carboxyl, amino (—NH₂), isocyanate (—NCO),isothiocyanate (—NCS), hydrazine (—NHNH₂), semicarbazide (—NHCONHNH₂),thiosemicarbazide (—NHCSNHNH₂), chloroacetamide (—NHCOCH₂Cl),bromoacetamide (—NHCOCH₂Br), iodoacetamide (—NHCOCH₂I), acylamino, suchas, for example acetylamino (—NHCOCH₃), mixed anhydrides, azide,hydroxide, sulfonyl chloride, carbodiimide or a group of formulas

in which Hal represents a halogen atom, is suitable.

Activated carboxyl groups are defined above as those carboxyl groupsthat can be derivatized in such a way that they facilitate the reactionwith a biomolecule. Which groups can be used for activation is known,and reference can be made to, for example, M. and A. Bodanszky, “ThePractice of Peptide Synthesis,” Springerverlag 1984. Examples are aductsof carboxylic acid with carbodiimides or activated esters, such as,e.g., hydroxybenzotriazole esters. Especially preferred is the activatedcarboxyl group for X that is selected from

In formula I, Z stands for a hydrogen atom or a metal ion equivalent.Which metal ion in the compound according to the invention is to becomplexed here depends on the intended use of the conjugates that areproduced with the inventions according to the invention with abiomolecule. Corresponding conjugates are suitable, for example, for NMRdiagnosis, radiodiagnosis and radiotherapy and neutron capture therapy.The conjugates in NMR diagnosis are especially preferably used ascontrast media.

The production of complexes for NMR diagnosis can be carried out as wasdisclosed in Patents EP 71564, EP 130934 and DE-OS 34 01 052. To thisend, the metal oxide or a metal salt (for example a chloride, nitrate,acetate, carbonate or sulfate) of the desired element is dissolved orsuspended in water and/or a lower alcohol (such as methanol, ethanol orisopropanol) and reacted with the solution or suspension of theequivalent amount of the complexing agent according to the invention.

If the complexing agents are to be used for the production ofradiodiagnostic agents or radiotherapeutic agents, the production of thecomplexes from the complexing agents can be carried out according to themethods that are described in “Radiotracers for Medical Applications,”Vol. I, CRC Press, Boca Raton, Fla.

The compounds according to the invention are used

-   -   1. For NMR diagnosis in the form of their complexes with the        ions of the paramagnetic elements with atomic numbers 21-29, 42,        44 and 58-70.

Suitable ions are, for example, the chromium(III), ion(II), cobalt(II),nickel(II), copper(II), praseodymium(III), neodymium(III), samarium(III)and ytterbium(III) ion. Because of their strong magnetic moment, thegadolinum(II), terbium(III), dysprosium(III), holmium(III), erbium(III),manganese(II) and iron(III) ions are especially preferred for NMRdiagnosis.

-   -   2. For radiodiagnosis and radiotherapy in the form of their        complexes with the radioisotopes of elements with atomic numbers        26, 27, 29, 31, 32, 37-39, 43, 46, 47, 49, 61, 62, 64, 67, 70,        71, 75, 77, 82 and 83.

The compounds according to the invention and especially their conjugateswith biomolecules meet the many different requirements for suitabilityas contrast media for nuclear spin tomography. After oral or parenteraladministration, they are thus extremely well suited for enhancing theinformational value of the image that is obtained with the aid of anuclear spin tomograph by increasing the signal intensity. They alsoshow the high effectiveness that is necessary to load the body with thesmallest possible amounts of foreign substances and the goodcompatibility that is necessary to maintain the non-invasive nature ofthe studies.

The good water solubility and low osmolality of the compounds accordingto the invention and their conjugates with biomolecules allow for theproduction of highly concentrated solutions so as to keep the volumeburden of the circulatory system within reasonable limits and to offsetthe dilution by bodily fluids, i.e., NMR diagnostic agents have to be100 to 1000 times more water-soluble than for NMR spectroscopy. Inaddition, the compounds according to the invention have not only a highstability in vitro but also a surprisingly high stability in vivo, sothat a release or an exchange of the ions, which are inherently toxicand not covalently bonded in the complexes, is carried out onlyextremely slowly within the time that it takes for the new contrastmedia to be completely excreted again.

The complex compounds according to the invention can also be usedadvantageously as susceptibility reagents and as shift reagents for invivo NMR spectroscopy.

The compounds according to the invention and their conjugates withbiomolecules are also suitable as radiodiagnostic agents andradiotherapeutic agents based on their advantageous radioactiveproperties and the good stability of the complex compounds that arecontained therein. Details of their use and dosage are described in,e.g., “Radiotracers for Medical Applications,” CRC Press, Boca Raton,Fla. 1983, as well as in Eur. J. Nucl. Med. 17 (1990) 346-364 and Chem.Rev. 93 (1993) 1137-1156.

For SPECT, the complexes with isotopes ¹¹¹In and ^(99m)Tc are suitable.

Another imaging method with radioisotopes is the positron-emissiontomography, which uses positron-emitting isotopes such as, e.g., ⁴³Sc,⁴⁴Sc, ⁵²Fe, ⁵⁵Co, ⁶⁸Ga, ⁶⁴Cu, ⁸⁶Y and ^(94m)Tc (Heiss, W. D.; Phelps, M.E.; Positron Emission Tomography of Brain, Springer Verlag Berlin,Heidelberg, New York 1983).

The compounds according to the invention and their conjugates withbiomolecules are also suitable, surprisingly enough, for differentiatingmalignant and benign tumors in areas without blood-brain barriers.

They are distinguished in that they are completely eliminated from thebody and thus are well-tolerated.

Since the compounds according to the invention and especially theirconjugates with biomolecules accumulate in malignant tumors (nodiffusion in healthy tissue, but high permeability of tumor vessels),they can also support the radiation therapy of malignant tumors. Thelatter is distinguished from the corresponding diagnosis only by theamount and type of the isotope that is used. The purpose in this case isthe destruction of tumor cells by high-energy short-wave radiation withthe lowest possible range of action. For this purpose, interactions ofthe metals that are contained in the complexes (such as, e.g., iron orgadolinium) with ionizing radiations (e.g., x rays) or with neutron raysare employed. By this effect, the local radiation dose at the site wherethe metal complex is found (e.g., in tumors) increases significantly. Toproduce the same radiation dose in the malignant tissue, radiationexposure for healthy tissue can be considerably reduced and thusburdensome side effects for the patients can be avoided when such metalcomplexes are used. The metal complex conjugates according to theinvention are therefore also suitable as radio-sensitizing substances inthe radiation therapy of malignant tumors (e.g., exploiting Mossbauereffects or neutron capture therapy). Suitable β-emitting ions are, e.g.,⁴⁶Sc, ⁴⁷Sc, ⁴⁸Sc, ⁷²Ga, ⁷³Ga, ⁹⁰Y, ⁶⁷Cu, ¹⁰⁹Pd, ¹¹¹Ag, ¹⁴⁹Pm, ¹⁵³Sm,¹⁶⁶Ho, ¹⁷⁷Lu, ¹⁸⁶ Re and ¹⁸⁸Re ⁹⁰Y, ¹⁷⁷Lu, ⁷²Ga, ¹⁵³Sm and ⁶⁷Cu arepreferred. Suitable α-emitting ions that have short half-lives are,e.g., ²¹¹At, ²¹¹Bi, ²¹²Bi, ²¹³Bi and ²¹⁴Bi, whereby ²¹²Bi is preferred.A suitable photon- and electron-emitting ion is ¹⁵⁸Gd, which can beobtained from ¹⁵⁷Gd by neutron capture.

If the compound according to the invention or conjugate thereof with abiomolecule is intended for use in the variant of the radiation therapythat is proposed by R. L. Mills et al. [Nature Vol. 336 (1988), p. 787],the central ion must be derived from a Mössbauer isotope, such as, forexample, ⁵⁷Fe or ¹⁵¹Eu.

The neutralization of optionally still present free carboxy groups iscarried out with the aid of inorganic bases (e.g., hydroxides,carbonates or bicarbonates) of, e.g., sodium, potassium, lithium,magnesium or calcium and/or organic bases, such as, i.a., primary,secondary and tertiary amines, such as, e.g., ethanolamine, morpholine,glucamine, N-methylglucamine and N,N-dimethylglucamine, as well as basicamino acids, such as, e.g., lysine, arginine and ornithine or amides oforiginally neutral or acidic amino acids.

For the production of natural complex compounds, as much of the desiredbase can be added, for example, into acid complex salts in aqueoussolution or suspension so that the neutral point is reached. Thesolution that is obtained can then be evaporated to the dry state in avacuum. It is often advantageous to precipitate the neutral salts thatare formed by adding water-miscible solvents, such as, e.g., loweralcohols (methanol, ethanol, isopropanol, etc.), lower ketones (acetone,etc.), polar ethers (tetrahydrofuran, dioxane, 1,2-dimethoyethane, etc.)and thus to obtain easily isolated and readily purified crystallizates.It has proven especially advantageous to add the desired base as earlyas during the complexing of the reaction mixture and thus to save aprocess step.

The compounds of formula I according to the invention can be producedaccording to the process that is known to one skilled in the art. Forexample, the compounds of formula I can be obtained by a process inwhich a compound of formula II

in which B¹, B², B³ and B⁴ are defined as above is optionally reactedafter introducing protective groups for the nitrogen atoms with Nu-A-X′and Nu-CH(R¹)—CO₂Z′, Nu-CH(R²)—CO₂Z′ and Nu-CH(R³)—CO₂Z′, whereby A andR¹, R², and R³ are defined as above and Nu is a nucleofuge, X′ standsfor X or a protected form of X, and X is defined as above and Z′ standsfor a hydrogen atom, a metal ion equivalent, preferably an alkali metalor alkaline-earth metal, such as especially sodium or potassium, or aprotective group for carboxyl. Then, the optionally present protectivegroups can be removed, and it can be reacted in a way that is known inthe art with at least one metal oxide or metal salt of a desiredelement. Then, in the thus obtained complexes, still present acidhydrogen atoms optionally can be substituted completely or partially bycations of inorganic and/or organic bases, amino acids or amino acidamides.

Three preferred process variants are described in more detail below.

Further process variants, especially where R¹, R² and R³ do notrepresent the same radicals, are shown in the examples. The describedprocess variants do not limit the extend of the invention.

In the compounds according to the invention, B¹, B², B³ and B⁴ canrepresent the same residues, whereas R¹, R², and/or R³ can representdifferent residues. Furthermore B¹, B², B³ and B⁴ can representdifferent residues in the molecule and R¹, R², and/or R³ can representthe same residues. And in the compounds according to the invention B¹,B², B³ and B⁴ can represent different residues and also R¹, R², and/orR³ can represent different residues. Furthermore B¹, B², B³ and B⁴ canrepresent the same residues in the molecule and R¹, R², and/or R³ canalso represent the same residues.

In the first variant, the macrocyclic compound that is unsubstituted atthe nitrogens is first reacted with protected unit AX′. In this case,group A carries a nucleofuge as a leaving group. By stoichiometricreaction control, one of the four nitrogen atoms in the macrocycliccompound reacts with group A with the leaving group departing. In thisway, a monofunctionalized macrocyclic compound that contains radical Xin protected form (X′) is obtained. In the second reaction step, theremaining three nucleophilic nitrogen atoms of the macrocyclic compoundare reacted in each case with a protected carboxylic acid, which carriesa nucleofuge in α-position in the carboxyl group.

For example three equivalents of the protected carboxylic acids can beused in the second reaction step. It is possible to use one protectedcarboxylic acid. In this case, all residues R¹R² and R³ are the same.Alternatively up to three different protected carboxylic acids can beused in same process. In this case it is possible to introduce differentresidues R¹, R² and/or R³ into the molecule, wherein R¹, R² and R³ areas defined above. Of course the different protected carboxylic acidsshould add up to about 3 equivalents as a total. The different protectedcarboxylic acids can be used as a mixture, but it is preferred to addthe different protected carboxylic acids in different reaction steps.With this method it is possible to obtain a well defined chemicalcompound or a mixture of chemical compounds having differentsubstitution patterns. If a mixture of chemical compounds with differentsubstitution patterns is obtained, this mixture can be seperated byknown methods, such as chromatography, after the reaction. Suitablesynthetic approaches for obtaining chemical compounds, in which R¹, R²and R³ are different are shown in the examples and these exemplifiedprocesses can easily be adapted by a skilled person to obtain furthercompounds having different residues R¹, R² and/or R³. These and furthersynthetic approaches are known in the art and can employ steps of e.g.addition or cleavage of protecting groups in order to selectively choosethe substituents for each N-atom independently.After the protective groups are cleaved off from the carboxylic acidfunctionalities, the complex that consists of paramagnetic metal ionsand chelate ligands is finished by adding metal oxide or metal salt.This process variant is diagrammatically reproduced below, whereby theradicals in the formulas are defined as above:

In a second variant, a macrocyclic compound is used as an educt, whichcarries already suitable protective groups SG on three of the fournitrogen atoms. As protective groups, e.g., tert-butyl-oxycarbonyl(t-BOC), COCF₃, carbobenzoxy (Cbo) or fluorenyl-methoxycarbonyl (FMOC),etc. are suitable here. By the presence of the protective groups, onlyone of the four nitrogen atoms is nucleophilic and can react with A-X′,which for its part carries a nucleofuge Nu as in the variant above.After linkage of both molecules with the leaving group departing, acleavage of the three protective groups from the nitrogen atoms iscarried out. It follows the derivatization with the aid of thecarboxylic acid derivatives, as was already described for the variantsabove. This second process variant is diagrammatically reproduced below,whereby the radicals in the formulas are defined as above:

SG=Protective group (e.g., BOC, Cbo, COCF₃, FMOC, etc.)

In the third variant, first one of the four nitrogen atoms of themacrocyclic compound is blocked by a corresponding protective group SG.Examples of suitable protective groups are formyl, benzyl, boctrityl,etc. The reaction now is carried out on the three remaining nucleophilicnitrogen atoms with correspondingly protected carboxylic acidderivatives, which carry a corresponding nucleofuge in α-position. Forexample three equivalents of the protected carboxylic acids can be usedin the second reaction step. It is possible to use one protectedcarboxylic acid. In this case, all residues R¹, R² and R³ are the same.Alternatively up to three different protected carboxylic acids can beused in same process. In this case it is possible to introduce differentresidues R¹, R² and/or R³ into the molecule, wherein R¹, R² and R³ areas defined above. Of course the different protected carboxylic acidsshould add up to about 3 equivalents as a total. The different protectedcarboxylic acids can be used as a mixture, but it is preferred to addthe different protected carboxylic acids in different reaction steps.With this method it is possible to obtain a well defined chemicalcompound or a mixture of chemical compounds having differentsubstitution patterns. If a mixture of chemical compounds with differentsubstitution patterns is obtained, this mixture can be seperated byknown methods, such as chromatography, after the reaction. Suitablesynthetic approaches for obtaining chemical compounds, in which R¹, R²and R³ are different are shown in the examples and these exemplifiedprocesses can easily be adapted by a skilled person to obtain furthercompounds having different residues R¹, R² and/or R³. These and furthersynthetic approaches are known in the art and can employ steps of e.g.addition or cleavage of protecting groups in order to selectively choosethe substituents for each N-atom independently.Then, the cleavage of protective group SG that is first introduced atthe first nitrogen atom and derivatizing with AX′, which for its partalso carries a nucleofuge, are carried out. This third process variantis diagrammatically reproduced below, whereby the radicals in theformulas are defined as above:

Advantageously used as a nucleofuge are the radicals:

Cl, Br, I, O-triflate, mesylate and tosylate.

The reaction is performed in a mixture of water and organic solvents,such as: isopropanol, ethanol, methanol, butanol, dioxane,tetrahydrofuran, dimethylformamide, dimethyl acetamide, formamide ordichloromethane. Ternary mixtures that consist of water, isopropanol anddichloromethane are preferred.

The reaction is carried out in a temperature range of between −10° C.and 100° C., preferably between 0° C. and 30° C.

The protection of the above-named groups can be accomplished in numerousways that are familiar to one skilled in the art. The embodiments thatare described below are used to explain these protective grouptechniques without being limited to these synthesis methods.

As acid protective groups, C₁-C₆-alkyl, C₆-C₁₀-aryl andC₆-C₁₀—Ar(C₁-C₄)-alkyl groups as well as trialkylsilyl groups aresuitable. The methyl, ethyl, propyl, isopropyl, n-butyl, i-butyl andtert-butyl groups are preferred.

The cleavage of these acid protective groups is carried out according tothe processes that are known to one skilled in the art, for example byhydrolysis, hydrogenolysis, alkaline saponification of the esters withalkali in aqueous-alcoholic solution at temperatures from 0 to 50° C.,acidic saponification with mineral acids or in the case of tert-butylesters with the aid of trifluoroacetic acid.

The NH groups can be protected in a variety of ways and then exposedagain. The N-trifluoroacetyl derivative is cleaved by potassium orsodium carbonate in water (H. Newman, J. Org. Chem., 30: 287 (1965), M.A. Schwartz et al., J. Am. Chem. Soc., 95 G12 (1973)) or simply byammonia solution (M. Imazama and F. Eckstein, J. Org. Chem., 44: 2039(1979)). The tert-butyloxycarbonyl derivative is equally easy to cleave:stirring with trifluoroacetic acid suffices (B. F. Lundt et al., J. Org.Chem., 43: 2285 (1978)). The group of NH protective groups to be cleavedhydrogenolytically or in a reductive manner is very large: the N-benzylgroup can be cleaved easily with hydrogen/Pd—C (W. H. Hartung and R.Rimonoff, Org. Reactions VII, 262 (1953)), which also applies for thetrityl group (L. Zervas et al., J. Am. Chem. Soc., 78; 1359 (1956)) andthe benzyloxycarbonyl group (M. Bergmann and L. Zervas Ber. 65: 1192(1932)).

The activated esters of the above-described compounds are produced asknown to one skilled in the art. For the case of isothiocyanates orα-haloacetates, the corresponding terminal amino precursors are reactedaccording to methods that are known in the literature with thiophosgeneor 2-halo-acetic acid-halides. The reaction with correspondinglyderivatized esters of N-hydroxysuccinimide, such as, for example:

is also possible (Hal=halogen).

In general, for this purpose, all commonly used activation methods forcarboxylic acids that are known in the prior art can be used. Themolecule Nu-A-X is preferably synthesized first independently. If themolecule contains an amide group, the latter is produced, for example,by an activated carboxylic acid being reacted with an amine. Theactivation of the carboxylic acid is carried out according to thecommonly used methods. Examples of suitable activating reagents aredicyclohexylcarbodiimide (DCC),1-ethyl-3-(3-dimethylaminopropyl)-carbodiimide-hydrochloride (EDC),benzotriazol-1-yloxytris-(dimethylamino)-phosphonium hexafluorophosphate(BOP) andO-(benzotriazol-1-yl)-1,1,3,3-tetramethyluroniumhexafluorophosphate(HBTU), preferably DCC. The addition of O-nucleophilic catalysts, suchas, e.g., N-hydroxysuccinimide (NHS) or N-hydroxybenzotriazole, is alsopossible.

If group X is a carboxylic acid function, the latter can be used inprotected form (e.g., in the form of benzyl ester), and the cleavage ofthe protective group can then be carried out hydrogenolytically.

To link this carboxylic acid function to a suitable functional group ofa suitable biomolecule, the latter should normally first be activated.Esters that are activated to this end are preferably produced at anintermediate stage, and said esters are then attacked by a nucleophilicgroup of the biomolecule. In this way, a covalent linkage between thebiomolecule and the compound of formula I according to the invention isproduced. Preferred activated esters are the esters of theN-hydroxysuccinimide, the esters of paranitrophenol or the esters ofpentafluorophenol. If group X in the form of an isothiocyanate is linkedto the biomolecule, a terminal amine is preferably first used which, ifnecessary, can be provided with a suitable protective group. Suitableprotective groups are known from peptide chemistry. After the protectivegroup is cleaved off, the isothiocyanate can be produced by reaction ofthe primary terminal amine with thiophosgene. Nucleophilic groups of thebiomolecule can be added to the latter.

In an embodiment, group X represents a maleinimide, which can react,e.g., selectively with thiol functions of the biomolecule.

In another embodiment, group X is a nucleophile (NH₂, SH), which affectsa suitable functionality of the biomolecule (activated ester,maleinimide, etc.). Numerous biomolecules that are functionalized withmaleinimides are commercially available.

This invention, moreover, relates to the use of the above-describedcompounds of formula I for the production of conjugates with abiomolecule.

The synthesis of the conjugates is generally carried out in such a waythat first a derivatized and functionalized chelate complex is producedthat then is linked to the biomolecule. It is also possible, however,that if synthetically produced biomolecules are used, the chelatecomplex according to the invention is incorporated in the latter duringthe synthesis of the biomolecule. This can be carried out, for example,during the sequential synthesis of oligopeptides in the synthesizingrobot. If necessary, the protective groups that are commonly used in thesynthesis of the corresponding biomolecule can be introduced into thecompound according to the invention. The latter are then cleaved againin the synthesizer in line with the usual synthesis algorithm.

“Biomolecule” is defined here as any molecule that either occurrednaturally, for example in the body, or was produced synthetically withan analogous structure. Moreover, among the latter, those molecules aredefined that can occur in interaction with a biological molecule thatoccurs, for example, in the body or a structure that occurs there, insuch a way, for example, that the conjugates accumulate at specificdesired spots of the body. “Body” is defined here as any plant or animalbody, whereby animal and especially human bodies are preferred.

Biomolecules are especially the molecules that occur in living creaturesthat as products of an evolutionary selection by orderly and complexinteractions meet specific objects of the organism and constitute thebasis of its vital functions (changes in material and shape,reproduction, energy balance). In biomolecules, simple building blocks(amino acids, nucleobases, monosaccharides, fatty acids, etc.) of largemolecules (proteins, nucleic acids, polysaccharides, lipids, etc.) areused in most cases. Corresponding macromolecules are also referred to asbiopolymers.

The biomolecule advantageously can have, for example, a polypeptideskeleton that consists of amino acids with side chains that canparticipate in a reaction with reactive group X of the compounds offormula I according to the invention. Such side chains include, forexample, the carboxyl groups of aspartic acid and glutamic acid esters,the amino groups of lysine radicals, the aromatic groups of tyrosine andhistidine radicals and the sulfhydryl groups of cysteine radicals.

A survey on biomolecules with numerous examples is found in themanuscript “Chemie der Biomoleküle [Chemistry of Biomolecules]” ofTU-Graz (H. Berthold et al., Institut für Organische Chemie [Institutefor Organic Chemistry], Tu-Graz, 2001), which can also be seen on theInternet under www.orgc.tu-graz.ac.at. The content of this document isintegrated by reference in this description.

To form conjugates with the compounds according to the invention, thefollowing biomolecules are especially suitable:

Biopolymers, proteins, such as proteins that have a biological function,HSA, BSA, etc., proteins and peptides, which accumulate at certain spotsin the organism (e.g., in receptors, cell membranes, at ducts, etc.),peptides that can be cleaved by proteases, peptides with predeterminedsynthetic sites of rupture (e.g., labile esters, amides, etc.), peptidesthat are cleaved by metalloproteases, peptides with photocleavablelinkers, peptides with oxidative agents (oxydases) and cleavable groups,peptides with natural and unnatural amino acids, glycoproteins(glycopeptides), signal proteins, antiviral proteins and apoctosis,synthetically modified biopolymers such as biopolymers that arederivatized with linkers, modified metalloproteases and derivatizedoxydase, etc., carbohydrates (mono- to polysaccharides), such asderivatized sugars, sugars that can be cleaved in the organism,cyclodextrins and derivatives thereof, amino sugars, chitosan,polysulfates and acetylneuraminic acid derivatives, antibodies, such asmonoclonal antibodies, antibody fragments, polyclonal antibodies,minibodies, single chains (also those that are linked by linkers tomultiple fragments), red blood corpuscles and other blood components,cancer markers (e.g., CAA) and cell adhesion substances (e.g., Lewis Xand anti-Lewis X derivatives), DNA and RNA fragments, such asderivatized DNAs and RNAs (e.g., those that were found by the SELEXprocess), synthetic RNA and DNA (also with unnatural bases), PNAs(Hoechst) and antisense, β-amino acids (Seebach), vector amines fortransfer into the cell, biogenic amines, pharmaceutical agents,oncological preparations, synthetic polymers, which are directed to abiological target (e.g., receptor), steroids (natural and modified),prostaglandins, taxol and derivatives thereof, endothelins, alkaloids,folic acid and derivatives thereof, bioactive lipids, fats, fatty acidesters, synthetically modified mono-, di- and triglycerides, liposomes,which are derivatized on the surface, micelles that consist of naturalfatty acids or perfluoroalkyl compounds, porphyrins, texaphrines,expanded porphyrins, cytochromes, inhibitors, neuramidases,neuropeptides, immunomodulators, such as FK 506, CAPE and gliotoxin,endoglycosidases, substrates that are activated by enzymes such ascalmodulin kinase, casein-kinase II, glutathione-S-transferase,heparinase, matrix-metalloproteases, β-insulin-receptor-kinase,UDP-galactose 4-epimerase, fucosidases, G-proteins, galactosidases,glycosidases, glycosyltransferases and xylosidase, antibiotics, vitaminsand vitamin analogs, hormones, DNA intercalators, nucleosides,nucleotides, lectins, vitamin B12, Lewis-X and related substances,psoralens, dienetriene antibiotics, carbacyclins, VEGF (vascularendothelial growth factor), somatostatin and derivatives thereof, biotinderivatives, antihormones, tumor-specific proteins and synthetic agents,polymers that accumulate in acidic or basic areas of the body(pH-controlled dispersion), myoglobins, apomyoglobins, etc.,neurotransmitter peptides, tumor necrosis factors, peptides thataccumulate in inflamed tissues, blood-pool reagents, anion andcation-transporter proteins, polyesters (e.g., lactic acid), polyamidesand polyphosphates.

Most of the above-mentioned biomolecules are commercially availablefrom, for example, Merck, Aldrich, Sigma, Calibochem or Bachem.

In addition, all “plasma protein binding groups” or “target bindinggroups” that are disclosed in WO 96/23526 and WO 01/08712 can be used asbiomolecules. The content of these two laid-open specifications istherefore integrated by reference into this description.

The number of compounds of formula I according to the invention perbiomolecule is random in principle, but a molecular ratio of 0.1:1 to10:1, especially 0.5:1 to 7:1, is preferred.

The compounds according to the invention are also suitable forconjugation on all molecules that are reacted with fluorescence dyes inthe prior art to determine, for example, their location byepifluorescence microscopy within the cell. After the administration ofthe medication, the compounds with, in principle, any medications canalso be conjugated to then track the transport within the organism, forexample by the NMR technique. It is also possible that the conjugatesfrom the compounds according to the invention and the biomoleculescontain other additional molecules, which had been conjugated on thebiomolecules. The term “biomolecule” in terms of this invention thusencompasses all molecules that occur in the biological systems and allmolecules that are biocompatible.

The conjugates that are obtained with the compounds according to theinvention are preferably used as contrast media in NMR diagnosis. Theconjugates should therefore be water-soluble. If the conjugates that areobtained with the compounds according to the invention are to be used asNMR contrast media, they are preferably dosed in an amount of 0.0001-5mmol/kg of body weight and especially preferably in an amount of0.005-0.5 mmol/kg of body weight. Details of use are discussed in, e.g.,H.-J. Weinmann et al., Am. J. of Roentgenology 142, 619 (1984). By thesurprisingly high relaxivity of the compounds according to the inventionwith simultaneous target specificity of the conjugates that are obtainedwith these compounds, the latter can be especially low-dosed, forexample, to detect tumors.

Details of use of radiotherapeutic agents are discussed in, e.g., R. W.Kozak et al. TIBTEC, October 1986, 262 (see above Bioconjugate Chem. 12(2001) 7-34).

This invention is explained in more detail by the examples below withoutbeing limited thereto.

Without further elaboration, it is believed that one skilled in the artcan, using the preceding description, utilize the present invention toits fullest extent. The following preferred specific embodiments are,therefore, to be construed as merely illustrative, and not limitative ofthe remainder of the disclosure in any way whatsoever.

In the foregoing and in the following examples, all temperatures are setforth uncorrected in degrees Celsius and, all parts and percentages areby weight, unless otherwise indicated.

EXAMPLES Example 1 a)10-[4-(Benzyloxycarbonyl)-1-methyl-2-oxo-3-azabutyl]-1,4,7-α,α′,α″-trimethyl-1,4,7-tris-(benzyloxycarbonylmethyl)-1,4,7,10-tetraazacyclododecane

25 g (81.1 mmol) of 2-bromopropionylglycine-benzyl ester (Example 1e ofWO 98/24774) is added to 27.9 g (162.2 mol) of1,4,7,10-tetraazacyclododecane, dissolved in 300 ml of chloroform, andit is stirred overnight at room temperature. 250 ml of water is added,the organic phase is separated, and it is washed twice in each case with200 ml of water. The organic phase is dried on magnesium sulfate andevaporated to the dry state in a vacuum. The residue is chromatographedon silica gel (mobile solvent: chloroform/methanol/aqueous 25%ammonia=10/5/1). The thus obtained1-[4-(benzyloxycarbonyl)-1-methyl-2-oxo-3-azabutyl]-1,4,7,10-tetraazacyclododecane(19.6 g; 50 mmol; 62% of theory) and 60 ml (0.35 mol) ofN-ethyldiisopropylamine in 200 ml of dichloromethane are added to 62.45g (0.2 mol) of 2-(trifluoromethanesulfonyloxy)propanoic acid benzylester (Kitazaki et al., Chem. Pharm. Bull. (1999), 47(3), 360) in 400 mlof dichloromethane, and it is stirred for 6 hours under reflux and thenovernight at room temperature. It is extracted three times with 500 mlof water each, the organic phase is dried on magnesium sulfate andevaporated to the dry state. The residue is chromatographed on silicagel (mobile solvent: dichloromethane/methanol: 20/1). The fractions thatcontain the product are combined and concentrated by evaporation.

Yield: 32.0 g (73% of theory) of a colorless, crystalline powder

Elementary analysis:

Cld.: C, 68.39; H, 7.23; N, 7.98.

Fnd.: C, 67.95; H, 7.41; N, 8.22.

b)10-(4-Carboxy-1-methyl-2-oxo-3-azabutyl)-1,4,7-α,α′,α″-trimethyl-1,4,7-tris(carboxy-methyl)-1,4,7,10-tetraazacyclododecane

26.3 g (30 mmol) of the title compound of Example 1a is dissolved in 400ml of isopropanol, mixed with 40 ml of water, and 3 g of palladiumcatalyst (10% Pd/C) is added. It is hydrogenated for 8 hours at 50° C.Catalyst is filtered out, and the filtrate is evaporated to the drystate in a vacuum.

Yield: 15.7 g (quantitative) of a colorless powder

Elementary analysis:

Cld.: C, 51.05; H, 7.60; N, 13.53.

Fnd.: C, 50.71; H, 7.83; N, 13.25.

c) Gd Complex of10-(4-carboxy-1-methyl-2-oxo-3-azabutyl)-1,4,7-α,α′,α″-trimethyl-1,4,7-tris(carboxymethyl)-1,4,7,10-tetraazacyclododecane

10.4 g (20 mmol) of the ligand that is described in Example 1b isdissolved in 200 ml of water and 80 ml of isopropanol, and it isacidified by adding 5 ml of acetic acid. 3.6 g (10 mmol) of gadoliniumoxide is added, and it is refluxed for 3 hours. After complexing iscompleted, it is set at pH 7.4 again with ammonia and chromatographed onsilica gel (mobile solvent: dichloromethane/methanol/ammonia: 20/20:1).The fractions that contain the product are combined and added via anIR-120® cation exchange column (H⁺ form). The acidic eluate isfreeze-dried.

Yield: 10.1 g (69% of theory) of a colorless powder.

Water content (Karl-Fischer): 8.3%

Elementary analysis (relative to anhydrous substance):

Cld.: C, 39.33; H, 5.40; Gd, 23.41; N, 10.42.

Fnd.: C, 39.21; H, 5.88; Gd, 22.93; N, 10.11.

Example 2 a)10-[4-(Benzyloxycarbonyl)-1-methyl-2-oxo-3-azabutyl]-1,4,7-α,α′,α″-tris(isopropyl)-1,4,7-tris(benzyloxycarbonylmethyl)-1,4,7,10-tetraazacyclododecane

19.6 g (50 mmol) of the1-[4-(benzyloxy-carbonyl)-1-methyl-2-oxo-3-azabutyl]-1,4,7,10-tetraazacyclododecanethat is described in Example 1a as an intermediate product and 60 ml(0.35 mol) of N-ethyldiisopropylamine in 200 ml of dichloromethane areadded to 68.1 g (0.2 mol) of 2-(trifluoromethanesulfonyloxy)-isovalericacid benzyl ester (Walker et al., Tetrahedron (1997), 53(43), 14591) in400 ml of dichloromethane, and it is stirred for 6 hours under refluxand then overnight at room temperature. It is extracted three times with500 ml of water each, the organic phase is dried on magnesium sulfateand evaporated to the dry state. The residue is chromatographed onsilica gel (mobile solvent: dichloromethane/methanol: 20/1). Thefractions that contain the product are combined and concentrated byevaporation.

Yield: 33.7 g (70% of theory) of a colorless, crystalline powder

Elementary analysis:

Cld.: C, 69.90; H, 7.86; N, 7.28.

Fnd.: C, 69.77; H, 7.51; N, 7.22.

b)10-(4-Carboxy-1-methyl-2-oxo-3-azabutyl)-1,4,7-α,α′,α″-tris(isopropyl)-1,4,7-tris(carboxymethyl)-1,4,7,10-tetraazacyclododecane

28.9 g (30 mmol) of the title compound of Example 2a is dissolved in 400ml of isopropanol, mixed with 40 ml of water, and 3 g of palladiumcatalyst (10% Pd/C) is added. It is hydrogenated for 8 hours at 50° C.Catalyst is filtered out, and the filtrate is evaporated to the drystate in a vacuum.

Yield: 18.0 g (quantitative) of a colorless powder

Elementary analysis:

Cld.: C, 55.89; H, 8.54; N, 11.64.

Fnd.: C, 55.63; H, 8.83; N, 11.31.

c) Gd Complex of10-(4-carboxy-1-methyl-2-oxo-3-azabutyl)-1,4,7-α,α′,α″-tris(isopropyl)-1,4,7-tris(carboxymethyl)-1,4,7,10-tetraazacyclododecane

12.0 g (20 mmol) of the ligand that is described in Example 2b isdissolved in 200 ml of water and 80 ml of isopropanol and acidified byadding 5 ml of acetic acid. 3.6 g (10 mmol) of gadolinium oxide isadded, and it is refluxed for 3 hours. After complexing is completed, itis set at pH 7.4 with ammonia and chromatographed on silica gel (mobilesolvent: dichloromethane/methanol/ammonia: 20/20/1). The fractions thatcontain the product are combined and added via an IR-120® cationexchange column (H⁺ form). The acidic eluate is freeze-dried.

Yield: 12.0 g (72% of theory) of a colorless powder.

Water content (Karl-Fischer): 9.1%

Elementary analysis (relative to anhydrous substance):

Cld.: C, 44.49; H, 6.40; Gd, 20.80; N, 9.26.

Fnd.: C, 44.21; H, 6.72; Gd, 20.23; N, 9.11.

Example 3 a)10-[4-(Benzyloxycarbonyl)-1-methyl-2-oxo-3-azabutyl]-1,4,7-α,α′,α″-tris(cyclohexyl)-1,4,7-tris(benzyloxycarbonylmethyl)-1,4,7,10-tetraazacyclododecane

19.6 g (50 mmol) of1-[4-(benzyloxy-carbonyl)-1-methyl-2-oxo-3-azabutyl]-1,4,7,10-tetraazacyclododecanethat is described in Example 1a as an intermediate product and 60 ml(0.35 mol) of N-ethyldiisopropylamine in 200 ml of dichloromethane areadded to 76.1 g (0.2 mol) of2-(trifluoromethanesulfonyloxy)-2-cyclohexylacetic acid benzyl ester(Qabar et al., Tetrahedron Letters (1998), 39(33), 5895) in 400 ml ofdichloromethane, and it is stirred for 6 hours under reflux and thenovernight at room temperature. It is extracted three times with 500 mlof water each, the organic phase is dried on magnesium sulfate andevaporated to the dry state. The residue is chromatographed on silicagel (mobile solvent: dichloromethane/methanol: 20/1). The fractions thatcontain the product are combined and concentrated by evaporation.

Yield: 41.1 g (76% of theory) of a colorless, crystalline powder

Elementary analysis:

Cld.: C, 72.13; H, 8.10; N, 6.47.

Fnd.: C, 71.88; H, 8.21; N, 6.25.

b)10-(4-Carboxy-1-methyl-2-oxo-3-azabutyl)-1,4,7-α,α′,α″-tris(cyclohexyl)-1,4,7-tris(carboxymethyl)-1,4,7,10-tetraazacyclododecane

32.5 g (30 mmol) of the title compound of Example 3a is dissolved in 400ml of isopropanol, mixed with 40 ml of water, and 3 g of palladiumcatalyst (10% Pd/C) is added. It is hydrogenated for 8 hours at 50° C.Catalyst is filtered out, and the filtrate is evaporated to the drystate in a vacuum.

Yield: 22.0 g (quantitative) of a colorless powder

Elementary analysis:

Cld.: C, 61.56; H, 8.80; N, 9.70.

Fnd.: C, 61.17; H, 8.98; N, 9.41.

c) Gd Complex of the10-(4-carboxy-1-methyl-2-oxo-3-azabutyl)-1,4,7-α,α′,α″-tris(cyclohexyl)-1,4,7-tris(carboxymethyl)-1,4,7,10-tetraazacyclododecane

14.4 g (20 mmol) of the ligand that is described in Example 3b isdissolved in 150 ml of water and 150 ml of isopropanol and acidified byadding 5 ml of acetic acid. 3.6 g (10 mmol) of gadolinium oxide isadded, and it is refluxed for 8 hours. After complexing is completed, itis set at pH 7.4 again with ammonia and chromatographed on silica gel(mobile solvent: dichloromethane/methanol/ammonia: 20/20/1). Thefractions that contain the product are combined and evaporated to thedry state. The residue is taken up with formic acid and evaporated tothe dry state several times with the addition of dichloromethane andthen dried in a vacuum until a constant weight is reached.

Yield: 12.4 g (65% of theory) of a colorless powder.

Water content (Karl-Fischer): 8.0%

Elementary analysis (relative to anhydrous substance):

Cld.: C, 50.72; H, 6.90; Gd 17.95; N, 7.99.

Fnd.: C, 51.03; H, 7.08; Gd, 17.42; N, 8.11.

Example 4 a)10-[4-(t-Butoxycarbonyl)-1-phenyl-2-oxo-3-azabutyl]-1,4,7-α,α′,α″-trimethyl-1,4,7-tris-(benzyloxycarbonylmethyl)-1,4,7,10-tetraazacyclododecane

26.6 g (81.1 mmol) of N-[2-bromo-2-phenylacetyl]-glycine-t-butylester(Example 6a of WO 98/24775) is added to 27.9 g (162.2 mmol) of1,4,7,10-tetraazacyclododecane, dissolved in 300 ml of chloroform, andit is stirred overnight at room temperature. 250 ml of water is added,the organic phase is separated, and it is washed twice in each case with200 ml of water. The organic phase is dried on magnesium sulfate andevaporated to the dry state in a vacuum. The residue is chromatographedon silica gel (mobile solvent: chloroform/methanol/aqueous 25%ammonia=10/5/1). The thus obtained1-[4-(t-butoxycarbonyl)-1-phenyl-2-oxo-3-azabutyl]-1,4,7,10-tetraazacyclododecane(21.0 g; 50 mmol; 62% of theory) and 60 ml (0.35 mol) ofN-ethyldiisopropylamine in 200 ml of dichloromethane are added to 62.45g (0.2 mol) of 2-(trifluoromethanesulfonyl-oxy)propanoic acid benzylester (Kitazaki et al., Chem. Pharm. Bull. (1999), 47(3), 360) in 400 mlof dichloromethane, and it is stirred for 6 hours under reflux and thenovernight at room temperature. It is extracted three times with 500 mlof water each, the organic phase is dried on magnesium sulfate andevaporated to the dry state. The residue is chromatographed on silicagel (mobile solvent: dichloromethane/methanol: 20/1). The fractions thatcontain the product are combined and concentrated by evaporation.

Yield: 34.0 g (75% of theory) of a colorless, crystalline powder

Elementary analysis:

Cld.: C, 68.93; H, 7.45; N, 7.73.

Fnd.: C, 69.12; H, 7.57; N, 7.60.

b)10-(4-(t-Butyloxycarbonyl-1-phenyl-2-oxo-3-azabutyl)-1,4,7-α,α′,α″-trimethyl-1,4,7-tris(carboxy-methyl)-1,4,7,10-tetraazacyclododecane

27.2 g (30 mmol) of the title compound of Example 4a is dissolved in 400ml of isopropanol, mixed with 40 ml of water, and 3 g of palladiumcatalyst (10% Pd/C) is added. It is hydrogenated for 8 hours at 50° C.Catalyst is filtered out, and the filtrate is evaporated to the drystate in a vacuum.

Yield: 17.5 g (quantitative) of a colorless powder

Elementary analysis:

Cld.: C, 55.95; H, 7.13; N, 12.08.

Fnd.: C, 56.21; H, 6.99; N, 11.83.

c) Gd Complex of10-(4-carboxy-1-phenyl-2-oxo-3-azabutyl)-1,4,7-α,α′,α″-trimethyl-1,4,7-tris(carboxymethyl)-1,4,7,10-tetraazacyclododecane

11.6 g (20 mmol) of the t-butylester that is described in Example 4b isdissolved in a very little trifluoroacetic acid and stirred for 15minutes at room temperature. After 250 ml of diethyl ether is added, itis stirred for 2 more hours, the precipitate is suctioned off and driedin a vacuum. The thus obtained free ligand is dissolved in 200 ml ofwater and 80 ml of isopropanol, set at pH 7 with dilute ammonia andacidified by adding 5 ml of acetic acid. 3.6 g (10 mmol) of gadoliniumoxide is added, and it is refluxed for 3 hours. After complexing iscompleted, it is set at pH 7.4 again with ammonia and chromatographed onsilica gel (mobile solvent: dichloromethane/methanol/ammonia: 20/20/1).The fractions that contain the product are combined and added via anIR-120® cation exchange column (H⁺ form). The acidic eluate isfreeze-dried.

Yield: 11.6 g (72% of theory) of a colorless powder.

Water content (Karl-Fischer): 9.0%

Elementary analysis (relative to anhydrous substance):

Cld.: C, 44.19; H, 5.22; Gd 21.43; N, 9.54.

Fnd.: C, 43.91; H, 5.27; Gd, 21.09; N, 9.77.

Example 5 a) 4-(Ethoxycarbonylmethoxy)-phenylacetic acid methyl ester

10 g (60.2 mmol) of hydroxyphenylacetic acid methyl ester (Aldrich) isdissolved in 75 ml of acetone. 18.4 g (133 mmol) of solid potassiumcarbonate is added. 17.8 ml (123 mmol) of bromoacetic acid ethyl esteris added in drops under reflux within 15 minutes, it is kept at thistemperature for another 4 hours, and it is stirred overnight at roomtemperature. Precipitate is filtered out, the solution is evaporated tothe dry state and chromatographed on silica gel (hexane/ethyl acetate3:1). The fractions that contain the product are combined andconcentrated by evaporation.

Yield: 14.6 g (96% of theory)

Elementary analysis:

Cld.: C, 61.90; H, 6.39.

Fnd.: C, 61.67; H, 6.50.

b) α-Bromo-4-(ethoxycarbonylmethoxy)-phenylacetic acid methyl ester

13.5 g (53.5 mmol) of the title compound of Example 5a is dissolved in75 ml of carbon tetrachloride. 9.52 g (53.5 mmol) of N-bromosuccinimideand 48 mg of dibenzoyl peroxide are added, refluxed for 5 hours andstirred overnight at room temperature. The suspension is washed twicewith sodium bicarbonate solution and once with water, the organic phaseis dried with magnesium sulfate, desiccant is suctioned off, and thefiltrate is evaporated to the dry state in a vacuum. The residue ischromatographed on silica gel (hexane/ethyl acetate 3:1). The fractionsthat contain the product are combined and concentrated by evaporation.

Yield: 15.4 g (87% of theory)

Elementary analysis:

Cld.: C, 47.15; H, 4.57; Br, 24.13.

Fnd.: C, 47.01; H, 4.76; Br, 23.70.

c)10-[α-(4-(Ethoxycarbonylmethoxy)phenyl)-methoxycarbonylmethyl]-1,4,7-α,α′,α″-trimethyl-1,4,7-tris(benzyloxycarbonylmethyl)-1,4,7,10-tetraazacyclododecane

26.9 g (81.1 mmol) of the bromine compound that is described in Example5b above is added to 27.9 g (162.2 mmol) of1,4,7,10-tetraazacyclododecane, dissolved in 300 ml of chloroform, andit is stirred overnight at room temperature. 250 ml of water is added,the organic phase is separated, and it is washed twice in each case with200 ml of water. The organic phase is dried on magnesium sulfate andevaporated to the dry state in a vacuum. The residue is chromatographedon silica gel (mobile solvent:dichloromethane/methanol/triethylamine=10/5/0.1). The thus obtained1-[α-(4-(ethoxycarbonylmethoxy)phenyl)-methoxycarbonylmethyl]-1,4,7,10-tetraazacyclododecane(21.1 g; 50 mmol; 62% of theory) and 60 ml (0.35 mol) ofN-ethyldiisopropylamine in 200 ml of dichloromethane are added to 62.45g (0.2 mol) of 2-(trifluoromethanesulfonyloxy)propanoic acid benzylester (Kitazaki et al., Chem. Pharm. Bull. (1999), 47(3), 360) in 400 mlof dichloromethane; and it is stirred for 6 hours under reflux and thenovernight at room temperature. It is extracted three times with 500 mlof water each, the organic phase is dried on magnesium sulfate andevaporated to the dry state. The residue is chromatographed on silicagel (mobile solvent: dichloromethane/methanol: 20/1). The fractions thatcontain the product are combined and concentrated by evaporation.

Yield: 34.1 g (75% of theory) of a colorless, crystalline powder

Elementary analysis:

Cld.: C, 67.38; H, 7.10; N, 6.16.

Fnd.: C, 67.20; H, 7.33; N, 6.31.

d)10-[α-(4-(Ethoxycarbonylmethoxy)phenyl)-methoxycarbonylmethyl]-1,4,7-α,α′,α″-trimethyl-1,4,7-tris(carboxymethyl)-1,4,7,10-tetraazacyclododecane

27.3 g (30 mmol) of the title compound of Example 5c is dissolved in 400ml of isopropanol, mixed with 40 ml of water, and 3 g of palladiumcatalyst (10% Pd/C) is added. It is hydrogenated for 8 hours at 50° C.Catalyst is filtered out, and the filtrate is evaporated to the drystate in a vacuum.

Yield: 19.3 g (quantitative) of a colorless powder

Elementary analysis:

Cld.: C, 56.42; H, 7.26; N, 8.77.

Fnd.: C, 56.21; H, 7.56; N, 8.47.

e) Gd Complex of10-[α-(4-carboxymethoxyphenyl)-carboxymethyl]-1,4,7-α,α′,α″-trimethyl-1,4,7-tris(carboxymethyl)-1,4,7,10-tetraazacyclododecane

13.3 g (20 mmol) of the title compound of Example 5d is taken up in 250ml of 2N sodium hydroxide solution and 250 ml of tetrahydrofuran, and itis stirred for 5 days at 40° C. Then, the aqueous phase is set at pH 7with Amberlite IR-120® (H⁺ form), 80 ml of isopropanol is added, and itis acidified by adding 5 ml of acetic acid. 3.6 g (10 mmol) of gadolinumoxide is added, and it is refluxed for 3 hours. After complexing iscompleted, it is set at pH 7.4 again with ammonia and chromatographed onsilica gel (mobile solvent: dichloromethane/methanol/ammonia: 20/20/1).The fractions that contain the product are combined and added via anIR-120® cation exchange column (H⁺ form). The acidic eluate isfreeze-dried.

Yield: 8.6 g (61% of theory) of a colorless powder.

Water content (Karl-Fischer): 9.3%

Elementary analysis (relative to anhydrous substance):

Cld.: C, 43.19; H, 4.97; Gd, 20.94; N, 7.46.

Fnd.: C, 43.22; H, 5.29; Gd, 20.42; N, 7.11.

Example 6 a) 4-(Ethoxycarbonylpropoxy)-phenylacetic acid methyl ester

10 g (60.2 mmol) of hydroxyphenylacetic acid methyl ester (Aldrich) isdissolved in 75 ml of acetone. 18.4 g (133 mmol) of solid potassiumcarbonate is added. 17.8 ml (123 mmol) of 4-bromobutyric acid ethylester is added in drops under reflux within 15 minutes, and it is keptat this temperature for another 4 hours and stirred overnight at roomtemperature. Precipitate is filtered out, the solution is evaporated tothe dry state, and it is chromatographed on silica gel (hexane/ethylacetate 3:1). The fractions that contain the product are combined andconcentrated by evaporation.

Yield: 16.4 g (97% of theory)

Elementary analysis:

Cld.: C, 64.27; H, 7.19.

Fnd.: C, 64.41; H, 6.92.

b) α-Bromo-[4-(ethoxycarbonylpropoxy)-phenyl]-acetic acid methyl ester

15.0 g (53.5 mmol) of the title compound of Example 6a is dissolved in75 ml of carbon tetrachloride. 9.52 g (53.5 mmol) of N-bromosuccinimideand 48 mg of dibenzoyl peroxide are added, and it is refluxed for 5hours and stirred overnight at room temperature. The suspension iswashed twice with sodium bicarbonate solution and once with water, theorganic phase is dried with magnesium sulfate, desiccant is filteredout, and the filtrate is evaporated to the dry state in a vacuum. Theresidue is chromatographed on silica gel (hexane/ethyl acetate 3:1). Thefractions that contain the product are combined and concentrated byevaporation.

Yield: 15.9 g (83% of theory)

Elementary analysis:

Cld.: C, 50.16; H, 5.33; Br, 22.24.

Fnd.: C, 50.33; H, 5.04; Br, 21.94.

c)10-[α-(4-(Ethoxycarbonylpropoxy)phenyl)-methoxycarbonylmethyl]-1,4,7-α,α′,α″-trimethyl-1,4,7-tris(benzyloxycarbonylmethyl)-1,4,7,10-tetraazacyclododecane

29.1 g (81.1 mmol) of the bromine compound that is described in Example6b above is added to 27.9 g (162.2 mmol) of1,4,7,10-tetraazacyclododecane, dissolved in 300 ml of chloroform, andit is stirred overnight at room temperature. 250 ml of water is added,the organic phase is separated, and it is washed twice in each case with200 ml of water. The organic phase is dried on magnesium sulfate andevaporated to the dry state in a vacuum. The residue is chromatographedon silica gel (mobile solvent:dichloromethane/methanol/triethylamine=10/5/0.1). The thus obtained1-[α-(4-(ethoxycarbonylpropoxy)phenyl)methoxycarbonylmethyl]-1,4,7,10-tetraazacyclododecane (22.5 g; 50 mmol; 62% of theory)and 60 ml (0.35 mol) of N-ethyldiisopropylamine in 200 ml ofdichloromethane are added to 62.45 g (0.2 mol) of2-(trifluoromethanesulfonyloxy)propanoic acid-benzyl ester (Kitazaki etal., Chem. Pharm. Bull. (1999), 47(3), 360) in 400 ml ofdichloromethane, and it is refluxed for 6 hours and then overnight atroom temperature. It is extracted three times with 500 ml each of water,the organic phase is dried on magnesium sulfate and evaporated to thedry state. The residue is chromatographed on silica gel (mobile solvent:dichloromethane/methanol: 20/1). The fractions that contain the productare combined and concentrated by evaporation.

Yield: 30.5 g (65% of theory) of a colorless, crystalline powder

Elementary analysis:

Cld.: C, 67.93; H, 7.31; N, 5.98.

Fnd.: C, 67.95; H, 7.22; N, 6.13.

d)10-[α-(4-(Ethoxycarbonylpropoxy)phenyl)-methoxycarbonylmethyl]-1,4,7-α,α′,α″-trimethyl-1,4,7-tris(carboxymethyl)-1,4,7,10-tetraazacyclododecane

28.1 g (30 mmol) of the title compound of Example 6c is dissolved in 400ml of isopropanol, mixed with 40 ml of water, and 3 g of palladiumcatalyst (10% Pd/C) is added. It is hydrogenated for 8 hours at 50° C.Catalyst is filtered out, and the filtrate is evaporated to the drystate in a vacuum.

Yield: 20.0 g (quantitative) of a colorless powder

Elementary analysis:

Cld.: C, 57.64; H, 7.56; N, 8.40.

Fnd.: C, 57.43; H, 7.77; N, 8.69.

e) Gd Complex of10-[α-(4-carboxypropoxyphenyl)-carboxymethyl]-1,4,7-α,α′,α″-trimethyl-1,4,7-tris(carboxymethyl)-1,4,7,10-tetraazacyclododecane

13.3 g (20 mmol) of the title compound of Example 6d is taken up in 250ml of 2N sodium hydroxide solution and 250 ml of tetrahydrofuran, and itis stirred for 5 days at 40° C. Then, the aqueous phase is set at pH 7with Amberlite IR-120® (H⁺ form), 80 ml of isopropanol is added, and itis acidified by adding 5 ml of acetic acid. 3.6 g (10 mmol) ofgadolinium oxide is added, and it is refluxed for 3 hours. Aftercomplexing is completed, it is set at pH 7.4 again with ammonia andchromatographed on silica gel (mobile solvent:dichloromethane/methanol/ammonia: 20/20/1). The fractions that containthe product are combined and added via an IR-120® cation exchange column(H⁺ form). The acidic eluate is freeze-dried.

Yield: 9.3 g (55% of theory) of a colorless powder.

Water content (Karl-Fischer): 8.0%

Elementary analysis (relative to anhydrous substance):

Cld.: C, 44.72; H, 5.31; Gd, 20.19; N, 7.19.

Fnd.: C, 44.31; H, 5.88; Gd, 19.93; N, 7.11.

Example 7 a) 4-(Ethoxycarbonyldecyloxy)-phenylacetic acid methyl ester

10 g (60.2 mmol) of hydroxyphenylacetic acid methyl ester (Aldrich) isdissolved in 75 ml of acetone. 18.4 g (133 mmol) of solid potassiumcarbonate is added, 36.1 g (123 mmol) of ω-bromoundecanoic acid ethylester in 50 ml of acetone is added in drops, refluxed for 8 hours andstirred overnight at room temperature. The undissolved material isfiltered out, the solution is evaporated to the dry state andchromatographed on silica gel (hexane/ethyl acetate 3:1). The fractionsthat contain the product are combined and concentrated by evaporation.

Yield: 20.3 g (89% of theory)

Elementary analysis:

Cld.: C, 69.81; H, 9.05.

Fnd.: C, 69.50; H, 8.91.

b) α-Bromo-[4-(ethoxycarbonyldecyloxy)-phenyl]-acetic acid methyl ester

20.2 g (53.5 mmol) of the title compound of Example 7a is dissolved in75 ml of carbon tetrachloride. 9.52 g (53.5 mmol) of N-bromosuccinimideand 48 mg of dibenzoyl peroxide are added, refluxed for 5 hours andstirred overnight at room temperature. The suspension is washed twicewith sodium bicarbonate solution and once with water, the organic phaseis dried with magnesium sulfate, desiccant is filtered out, and thefiltrate is evaporated to the dry state in a vacuum. The residue ischromatographed on silica gel (hexane/ethyl acetate 3:1). The fractionsthat contain the product are combined and concentrated by evaporation.

Yield: 21.0 g (86% of theory)

Elementary analysis:

Cld.: C, 57.77; H, 7.27; Br, 17.47.

Fnd.: C, 57.95; H, 7.41; Br, 17.02.

c)10-[α-(4-(Ethoxycarbonyldecyloxy)phenyl)-methoxycarbonylmethyl]-1,4,7-α,α′,α″-trimethyl-1,4,7-tris(benzyloxycarbonylmethyl)-1,4,7,10-tetraazacyclododecane

37.1 g (81.1 mmol) of the bromine compound that is described in Example7b above is added to 27.9 g (162.2 mmol) of1,4,7,10-tetraazacyclododecane, dissolved in 300 ml of chloroform, andit is stirred overnight at room temperature. 250 ml of water is added,the organic phase is separated, and it is washed twice in each case with200 ml of water. The organic phase is dried on magnesium sulfate andevaporated to the dry state in a vacuum. The residue is chromatographedon silica gel (mobile solvent:dichloromethane/methanol/triethylamine=10/5/0.1). The thus obtained1-[α-(4-(ethoxycarbonyldecyloxy)phenyl)-methoxycarbonylmethyl]-1,4,7,10-tetraazacyclododecane(27.4 g; 50 mmol; 62% of theory) and 60 ml (0.35 mol) ofN-ethyldiisopropylamine in 200 ml of dichloromethane are added to 62.45g (0.2 mmol) of 2-(trifluoromethanesulfonyloxy)propanoic acid benzylester (Kitazaki et al., Chem. Pharm. Bull. (1999), 47(3), 360) in 400 mlof dichloromethane, and it is stirred for 6 hours under reflux and thenovernight at room temperature. It is extracted three times with 500 mlof water each, the organic phase is dried on magnesium sulfate, and itis evaporated to the dry state. The residue is chromatographed on silicagel (mobile solvent: dichloromethane/methanol: 20/1). The fractions thatcontain the product are combined and concentrated by evaporation.

Yield: 33.6 g (65% of theory) of a colorless, crystalline powder

Elementary analysis:

Cld.: C, 69.61; H, 7.98; N, 5.41.

Fnd.: C, 69.75; H, 7.88; N, 5.12.

d)10-[α-(4-(Ethoxycarbonyldecyloxy)phenyl)-methoxycarbonylmethyl]-1,4,7-α,α′,α″-trimethyl-1,4,7-tris(carboxymethyl)-1,4,7,10-tetraazacyclododecane

31.1 g (30 mmol) of the title compound of Example 7c is dissolved in 400ml of isopropanol, mixed with 40 ml of water, and 3 g of palladiumcatalyst (10% Pd/C) is added. It is hydrogenated for 8 hours at 50° C.Catalyst is filtered out, and the filtrate is evaporated to the drystate in a vacuum.

Yield: 23.0 g (quantitative) of a colorless powder

Elementary analysis:

Cld.: C, 61.24; H, 8.43; N, 7.32.

Fnd.: C, 60.96; H, 8.61; N, 7.22.

e) Gd Complex of10-[α-(4-carboxydecyloxyphenyl)-carboxymethyl]-1,4,7-α,α′,α″-trimethyl-1,4,7-tris(carboxymethyl)-1,4,7,10-tetraazacyclododecane

15.3 g (20 mmol) of the title compound of Example 7d is taken up in 250ml of 2N sodium hydroxide solution and 250 ml of tetrahydrofuran, and itis stirred for 5 days at 40° C. Then, the aqueous phase is set at pH 7with Amberlite IR-120® (H⁺ form), 80 ml of isopropanol is added, and itis acidified by adding 5 ml of acetic acid. 3.6 g (10 mmol) ofgadolinium oxide is added, and it is refluxed for 3 hours. Aftercomplexing is completed, it is set at pH 7.4 again with ammonia, and itis chromatographed on silica gel (mobile solvent:dichloromethane/methanol/ammonia: 20/20/1). The fractions that containthe product are combined and added via an IR-120® cation exchange column(H⁺ form). The acidic eluate is freeze-dried.

Yield: 11.5 g (60% of theory) of a colorless powder.

Water content (Karl-Fischer): 8.5%

Elementary analysis (relative to anhydrous substance):

Cld.: C, 49.30; H, 6.32; Gd, 17.93; N, 6.39.

Fnd.: C, 49.56; H, 6.10; Gd, 17.52; N, 6.63.

Example 8 a)10-(p-Methoxycarbonylbenzyl)-1,4,7-α,α′,α″-trimethyl-1,4,7-tris(benzyloxycarbonyl-methyl)-1,4,7,10-tetraazacyclododecane

18.6 g (81.1 mmol) of 4-bromomethyl-benzoic acid methyl ester (Aldrich)in 150 ml of chloroform is added to 27.9 g (162.2 mmol) of1,4,7,10-tetraazacyclododecane, dissolved in 300 ml of chloroform, andit is stirred overnight at room temperature. 250 ml of water is added,the organic phase is separated, and it is washed twice in each case with200 ml of water. The organic phase is dried on magnesium sulfate andevaporated to the dry state in a vacuum. The residue is chromatographedon silica gel (mobile solvent: methanol/aqueous 25% ammonia=8/1). Thethus obtained 1-(p-methoxycarbonylbenzyl)-1,4,7,10-tetraazacyclododecane(21.6 g; 67.3 mmol; 83% of theory) and 60 ml (0.35 mol) ofN-ethyldiisopropylamine in 200 ml of dichloromethane are added to 62.45g (0.2 mol) of 2-(trifluoromethanesulfonyloxy)propanoic acid benzylester (Kitazaki et al., Chem. Pharm. Bull. (1999), 47(3), 360) in 400 mlof dichloromethane, and it is stirred for 6 hours under reflux and thenovernight at room temperature. It is extracted three times with 500 mlof water each, the organic phase is dried on magnesium sulfate andevaporated to the dry state. The residue is chromatographed on silicagel (mobile solvent: dichloromethane/methanol: 20/1). The fractions thatcontain the product are combined and concentrated by evaporation.

Yield: 41.8 g (77% of theory) of a colorless, crystalline powder

Elementary analysis:

Cld.: C, 69.95; H, 7.24; N, 6.94.

Fnd.: C, 69.57; H, 7.39; N, 7.12.

b)10-(p-Carboxybenzyl)-1,4,7-α,α′,α″-trimethyl-1,4,7-tris(carboxymethyl)-1,4,7,10-tetraazacyclododecane

24.2 g (30 mmol) of the title compound of Example 8a is dissolved in 400ml of methanol, mixed with 100 ml of 15N sodium hydroxide solution,refluxed for 6 hours and stirred overnight at room temperature. Afterconcentration by evaporation in a vacuum, the residue is dissolved in200 ml of water and set at pH 7 by adding IR-120® cation exchanger (H⁺form). Exchanger is filtered out and evaporated to the dry state in avacuum. The residue is complexed without being further characterized.

Thin-layer system: n-butanol/aqueous ammonia/ethanol/water 12/6/3/3

Yield: 16 g

c) Gd Complex of10-(p-carboxybenzyl)-1,4,7-α,α′,α″-trimethyl-1,4,7-tris(carboxymethyl)-1,4,7,10-tetraazacyclododecane

11 g (20 mmol) of the ligand that is described in Example 8b isdissolved in 200 ml of water and 80 ml of isopropanol and acidified byadding 5 ml of acetic acid. 3.6 g (10 mmol) of gadolinium oxide is addedand refluxed for 3 hours. After complexing is completed, it is set at pH7.4 again with ammonia and chromatographed on silica gel (mobilesolvent: dichloromethane/methanol/ammonia: 20/20/1). The fractions thatcontain the product are combined and added via an IR-120® cationexchange column (H⁺ form). The acidic eluate is freeze-dried.

Yield: 8.9 g (61% of theory) of a colorless powder.

Water content (Karl-Fischer): 7.2%

Elementary analysis (relative to anhydrous substance):

Cld.: C, 44.37; H, 5.21; Gd, 23.23; N, 8.28.

Fnd.: C, 44.12; H, 5.46; Gd, 22.93; N, 8.51.

Example 9 a)10-(p-Methoxycarbonylbenzyl)-1,4,7-α,α′,α″-tris(isopropyl)-1,4,7-tris(benzyloxycarbonyl-methyl)-1,4,7,10-tetraazacyclododecane

21.6 g (67.3 mmol) of the1-(p-methoxycarbonylbenzyl)-1,4,7,10-tetraazacyclododecane that isdescribed in Example 8a as an intermediate product and 60 ml (0.35 mol)of N-ethyldiisopropylamine in 200 ml of dichloromethane are added to85.1 g (0.25 mol) of 2-(trifluoromethanesulfonyloxy)-isovaleric acidbenzyl ester (Walker et al., Tetrahedron (1997), 53(43), 14591) in 400ml of dichloromethane, and it is stirred for 6 hours under reflux andthen overnight at room temperature. It is extracted three times with 500ml of water each, the organic phase is dried on magnesium sulfate andevaporated to the dry state. The residue is chromatographed on silicagel (mobile solvent: dichloromethane/methanol: 20/1). The fractions thatcontain the product are combined and concentrated by evaporation.

Yield: 48.5 g (81% of theory) of a colorless, crystalline powder

Elementary analysis:

Cld.: C, 71.43; H, 7.92; N, 6.29.

Fnd.: C, 71.12; H, 7.79; N, 6.55.

b)10-(p-Carboxybenzyl)-1,4,7-α,α′,α″-tris(isopropyl)-1,4,7-tris(carboxymethyl)-1,4,7,10-tetraazacyclododecane

26.7 g (30 mmol) of the title compound of Example 9a is dissolved in 400ml of methanol, mixed with 100 ml of 15N sodium hydroxide solution,refluxed for 6 hours and stirred overnight at room temperature. Afterconcentration by evaporation in a vacuum, the residue is dissolved in200 ml of water and set at pH 7 by adding IR-120® cation exchanger (H⁺form). Exchanger is filtered out, and it is evaporated to the dry statein a vacuum. The residue is complexed without being furthercharacterized.

Thin-layer system: n-butanol/aqueous ammonia/ethanol/water 12/6/3/3

Yield: 19 g

c) Gd Complex of10-(p-carboxybenzyl)-1,4,7-α,α′,α″-tris(isopropyl)-1,4,7-tris(carboxymethyl)-1,4,7,10-tetraazacyclododecane

12.6 g (20 mmol) of the ligand that is described in Example 9b isdissolved in 200 ml of water and 80 ml of isopropanol and acidified byadding 5 ml of acetic acid. 3.6 g (10 mmol) of gadolinium oxide isadded, and it is refluxed for 3 hours. After complexing is completed, itis set at pH 7.4 again with ammonia, and it is chromatographed on silicagel (mobile solvent: dichloromethane/methanol/ammonia: 20/20/1). Thefractions that contain the product are combined and added via an IR-120®cation exchange column (H⁺ form). The acidic eluate is freeze-dried.

Yield: 10.9 g (65% of theory) of a colorless powder.

Water content (Karl-Fischer): 9.0%

Elementary analysis (relative to anhydrous substance):

Cld.: C, 48.93; H, 6.23; Gd, 20.66; N, 7.36.

Fnd.: C, 48.87; H, 6.01; Gd, 20.22; N, 7.59.

Example 10 a)10-(p-Methoxycarbonylbenzyl)-1,4,7-α,α′,α″-tris(cyclohexyl)-1,4,7-tris(benzyloxycarbonyl-methyl)-1,4,7,10-tetraazacyclododecane

21.6 g (67.3 mmol) of the1-(p-methoxycarbonylbenzyl)-1,4,7,10-tetraazacyclododecane that isdescribed in Example 8a as an intermediate product and 60 ml (0.35 mol)of N-ethyldiisopropylamine in 200 ml of dichloromethane are added to95.1 g (0.25 mol) of 2-(trifluoromethanesulfonyloxy)-2-cyclohexylaceticacid benzyl ester (Qabar et al., Tetrahedron Letters (1998), 39(33),5895) in 400 ml of dichloromethane, and it is stirred for 6 hours underreflux and then overnight at room temperature. It is extracted threetimes with 500 ml of water each, the organic phase is dried on magnesiumsulfate and evaporated to the dry state. The residue is chromatographedon silica gel (mobile solvent: dichloromethane/methanol: 20/1). Thefractions that contain the product are combined and concentrated byevaporation.

Yield: 48.3 g (71% of theory) of a colorless, crystalline powder

Elementary analysis:

Cld.: C, 73.63; H, 8.17; N, 5.54.

Fnd.: C, 73.42; H, 8.39; N, 5.75.

b)10-(p-Carboxybenzyl)-1,4,7-α,α′,α″-tris(cyclohexyl)-1,4,7-tris(carboxymethyl)-1,4,7,10-tetraazacyclododecane

30.3 g (30 mmol) of the title compound of Example 10a is dissolved in400 ml of methanol, mixed with 100 ml of 15N sodium hydroxide solution,refluxed for 6 hours and stirred overnight at room temperature. Afterconcentration by evaporation in a vacuum, the residue is dissolved in200 ml of water and set at pH 7 by adding IR-120® cation exchanger (H⁺form). Exchanger is filtered out, and it is evaporated to the dry statein a vacuum. The residue is complexed without being furthercharacterized.

Thin-layer system: n-butanol/aqueous ammonia/ethanol/water 12/6/3/3

Yield: 22.5 g

c) Gd Complex of10-(p-carboxybenzyl)-1,4,7-α,α′,α″-tris(cyclohexyl)-1,4,7-tris(carboxymethyl)-1,4,7,10-tetraazacyclododecane

15.0 g (20 mmol) of the ligand that is described in Example 10b isdissolved in 200 ml of water and 80 ml of isopropanol and acidified byadding 5 ml of acetic acid. 3.6 g (10 mmol) of gadolinium oxide isadded, and it is refluxed for 3 hours. After complexing is completed, itis set at pH 7.4 again with ammonia and chromatographed on silica gel(mobile solvent: dichloromethane/methanol/ammonia: 20/20/1). Thefractions that contain the product are combined and evaporated to thedry state. The residue is taken up with formic acid and evaporated tothe dry state several times with the addition of dichloromethane andthen dried in a vacuum until a constant weight is reached.

Yield: 11.9 g (63% of theory) of a colorless powder.

Water content (Karl-Fischer): 7.0%

Elementary analysis (relative to anhydrous substance):

Cld.: C, 54.52; H, 6.75; Gd, 17.85; N, 6.36.

Fnd.: C, 54.19; H, 6.83; Gd, 17.61; N, 6.69.

Example 11 a)10-(p-Methoxycarbonylbenzyl)-1,4,7-α,α′,α″-triphenyl-1,4,7-tris(benzyloxycarbonyl-methyl)-1,4,7,10-tetraazacyclododecane

21.6 g (67.3 mmol) of1-(p-methoxycarbonylbenzyl)-1,4,7,10-tetraazacyclododecane that isdescribed in Example 8a as an intermediate product and 60 ml (0.35 mol)of N-ethyldiisopropylamine in 200 ml of dichloromethane are added to93.6 g (0.25 mol) of 2-(trifluoromethanesulfonyloxy)-2-phenylacetic acidbenzyl ester (Qabar et al., Tetrahedron Letters (1998), 39(33), 5895) in400 ml of dichloromethane, and it is stirred for 6 hours under refluxand then overnight at room temperature. It is extracted three times with500 ml of water each, the organic phase is dried on magnesium sulfateand evaporated to the dry state. The residue is chromatographed onsilica gel (mobile solvent: dichloromethane/methanol: 20/1). Thefractions that contain the product are combined and concentrated byevaporation.

Yield: 50.8 g (76% of theory) of a colorless, crystalline powder

Elementary analysis:

Cld.: C, 74.98; H, 6.49; N, 5.64.

Fnd.: C, 75.22; H, 6.61; N, 5.47.

b)10-(p-Carboxybenzyl)-1,4,7-α,α′,α″-triphenyl-1,4,7-tris(carboxymethyl)-1,4,7,10-tetraazacyclododecane

29.8 g (30 mmol) of the title compound of Example 11a is dissolved in400 ml of methanol, mixed with 100 ml of 15N sodium hydroxide solution,refluxed for 6 hours and stirred overnight at room temperature. Afterconcentration by evaporation in a vacuum, the residue is dissolved in200 ml of water and set at pH 7 by adding IR-120® cation exchanger (H⁺form). Exchanger is filtered out, and it is evaporated to the dry statein a vacuum. The residue is complexed without being furthercharacterized.

Thin-layer system: n-butanol/aqueous ammonia/ethanol/water 12/6/3/3

Yield: 22.0 g

c) Gd Complex of10-(p-carboxybenzyl)-1,4,7-α,α′,α″-triphenyl-1,4,7-tris(carboxymethyl)-1,4,7,10-tetraazacyclododecane

14.6 g (20 mmol) of the ligand that is described in Example 11b isdissolved in 200 ml of water and 80 ml of isopropanol, and it isacidified by adding 5 ml of acetic acid. 3.6 g (10 mmol) of gadoliniumoxide is added, and it is refluxed for 3 hours. After complexing iscompleted, it is set at pH 7.4 again with ammonia and chromatographed onsilica gel (mobile solvent: dichloromethane/methanol/ammonia: 20/20/1).The fractions that contain the product are combined and evaporated tothe dry state. The residue is taken up with formic acid and evaporatedto the dry state several times with the addition of dichloromethane andthen dried in a vacuum until a constant weight is reached.

Yield: 13.1 g (70% of theory) of a colorless powder.

Water content (Karl-Fischer): 8.1%

Elementary analysis (relative to anhydrous substance):

Cld.: C, 55.67; H, 4.79; Gd, 18.22; N, 6.49.

Fnd.: C, 55.33; H, 4.97; Gd, 17.92; N, 6.54.

Example 12 a)10-[4-(t-Butoxycarbonyl)-1-phenyl-2-oxo-3-azabutyl]-1,4,7-α,α′,α″-triphenyl-1,4,7-tris-(benzyloxycarbonylmethyl)-1,4,7,10-tetraazacyclododecane

26.6 g (81.1 mmol) of N-[2-bromo-2-phenylacetyl]-glycine-t-butylester(Example 6a of WO 98/24775) is added to 27.9 g (162.2 mmol) of1,4,7,10-tetraazacyclododecane, dissolved in 300 ml of chloroform, andit is stirred overnight at room temperature. 250 ml of water is added,the organic phase is separated, and it is washed twice in each case with200 ml of water. The organic phase is dried on magnesium sulfate andevaporated to the dry state in a vacuum. The residue is chromatographedon silica gel (mobile solvent: chloroform/methanol/aqueous 25%ammonia=10/5/1). The thus obtained1-[4-(t-butoxycarbonyl)-1-phenyl-2-oxo-3-azabutyl]-1,4,7,10-tetraazacyclododecane(21.0 g; 50 mmol; 62% of theory) and 60 ml (0.35 mol) ofN-ethyldiisopropylamine in 200 ml of dichloromethane are added to 74.9 g(0.2 mol) of 2-(trifluoromethanesulfonyloxy)-2-phenylacetic acid benzylester (Qabar et al., Tetrahedron Letters (1998), 39(33), 5895) in 400 mlof dichloromethane, and it is stirred for 6 hours under reflux and thenovernight at room temperature. It is extracted three times with 500 mlof water each, the organic phase is dried on magnesium sulfate andevaporated to the dry state. The residue is chromatographed on silicagel (mobile solvent: dichloromethane/methanol: 30/1). The fractions thatcontain the product are combined and concentrated by evaporation.

Yield: 37.7 g (69% of theory) of a colorless, crystalline powder

Elementary analysis:

Cld.: C, 73.67; H, 6.74; N, 6.41.

Fnd.: C, 73.44; H, 6.43; N, 6.79.

b)10-(4-(t-Butoxycarbonyl-1-phenyl-2-oxo-3-azabutyl)-1,4,7-α,α′,α″-triphenyl-1,4,7-tris(carboxy-methyl)-1,4,7,10-tetraazacyclododecane

32.8 g (30 mmol) of the title compound of Example 12a is dissolved in400 ml of isopropanol, mixed with 40 ml of water, and 3 g of palladiumcatalyst (10% Pd/C) is added. It is hydrogenated for 8 hours at 50° C.Catalyst is filtered out, and the filtrate is evaporated to the drystate in a vacuum.

Yield: 24.8 g (quantitative) of a colorless powder

Elementary analysis:

Cld.: C, 67.22; H, 6.74; N, 8.52.

Fnd.: C, 67.00; H, 6.85; N, 8.23.

c) Gd Complex of10-(4-carboxy-1-phenyl-2-oxo-3-azabutyl)-1,4,7-α,α′,α″-triphenyl-1,4,7-tris(carboxymethyl)-1,4,7,10-tetraazacyclododecane

16.4 g (20 mmol) of the t-butylester that is described in Example 12b isdissolved in very little trifluoroacetic acid, and it is stirred for 15minutes at room temperature. After 250 ml of diethyl ether is added, itis stirred for 2 more hours, the precipitate is suctioned off, and it isdried in a vacuum. The thus obtained free ligand is dissolved in 200 mlof water and 80 ml of isopropanol, set at pH 7 with dilute ammonia andacidified by adding 5 ml of acetic acid. 3.6 g (10 mmol) of gadoliniumoxide is added, and it is refluxed for 3 hours. After complexing iscompleted, it is set at pH 7.4 again with ammonia and chromatographed onsilica gel (mobile solvent: dichloromethane/methanol/ammonia: 25/15/1).The fractions that contain the product are combined and added via anIR-120® cation exchange column (H⁺ form). The acidic eluate isfreeze-dried.

Yield: 11.7 g (59% of theory) of a colorless powder.

Water content (Karl-Fischer): 7.5%

Elementary analysis (relative to anhydrous substance):

Cld.: C, 54.83; H, 4.82; Gd, 17.09; N, 7.61.

Fnd.: C, 54.91; H, 4.67; Gd, 16.62; N, 7.33.

Example 13 a)10-[4-(Benzyloxycarbonyl)-2-oxo-3-azabutyl]-1,4,7-α,α′,α″-tris(isopropyl)-1,4,7-tris(benzyloxy-carbonylmethyl)-1,4,7,10-tetraazacyclododecane

23.2 g (81.1 mmol) of 2-bromoacetylglycine-benzyl ester (Teger-Nilssonet al., WO 93/11152, page 38) is added to 34.4 g (0.2 mol) of1,4,7,10-tetraazacyclododecane, dissolved in 300 ml of chloroform, andit is stirred overnight at room temperature. 250 ml of water is added,the organic phase is separated, and it is washed twice in each case with200 ml of water. The organic phase is dried on magnesium sulfate andevaporated to the dry state in a vacuum. The residue is chromatographedon silica gel (mobile solvent: chloroform/methanol/aqueous 25%ammonia=10/5/1). The thus obtained1-[4-(benzyloxycarbonyl)-2-oxo-3-azabutyl]-1,4,7,10-tetraazacyclododecane(19.6 g; 50 mmol; 62% of theory) and 60 ml (0.35 mol) ofN-ethyldiisopropylamine in 200 ml of dichloromethane are added to 68.1 g(0.2 mol) of 2-(trifluoromethanesulfonyloxy)-isovaleric acid benzylester (Walker et al., Tetrahedron (1997), 53/43), 14591) in 400 ml ofdichloromethane, and it is stirred for 6 hours under reflux and thenovernight at room temperature. It is extracted three times with 500 mlof water each, the organic phase is dried on magnesium sulfate andevaporated to the dry state. The residue is chromatographed on silicagel (mobile solvent: dichloromethane/methanol: 20/1). The fractions thatcontain the product are combined and concentrated by evaporation.

Yield: 37.0 g (78% of theory) of a colorless, crystalline powder

Elementary analysis:

Cld.: C, 69.67; H, 7.76; N, 7.39.

Fnd.: C, 69.51; H, 7.88; N, 7.39.

b)10-(4-Carboxy-2-oxo-3-azabutyl)-1,4,7-α,α′,α″-tris(isopropyl)-1,4,7-tris(carboxymethyl)-1,4,7,10-tetraazacyclododecane

28.4 g (30 mmol) of the title compound of Example 13a is dissolved in400 ml of isopropanol, mixed with 40 ml of water, and 3 g of palladiumcatalyst (10% Pd/C) is added. It is hydrogenated for 8 hours at 50° C.Catalyst is filtered out, and the filtrate is evaporated to the drystate in a vacuum.

Yield: 17.7 g (quantitative) of a colorless powder

Elementary analysis:

Cld.: C, 55.18; H, 8.40; N, 11.92.

Fnd.: C, 54.97; H, 8.70; N, 11.88.

c) Gd Complex of10-(4-carboxy-2-oxo-3-azabutyl)-1,4,7-α,α′,α″-tris(isopropyl)-1,4,7-tris(carboxymethyl)-1,4,7,10-tetraazacyclododecane

11.8 g (20 mmol) of the ligand that is described in Example 13b isdissolved in 200 ml of water and 80 ml of isopropanol and acidified byadding 5 ml of acetic acid. 3.6 g (10 mmol) of gadolinium oxide isadded, and it is refluxed for 3 hours. After complexing is completed, itis set at pH 7.4 with ammonia and chromatographed on silica gel (mobilesolvent: dichloromethane/methanol/ammonia: 20/20/1). The fractions thatcontain the product are combined and added via an IR-120® cationexchange column (H⁺ form). The acidic eluate is freeze-dried.

Yield: 12.1 g (75% of theory) of a colorless powder.

Water content (Karl-Fischer): 8.0%

Elementary analysis (relative to anhydrous substance):

Cld.: C, 43.71; H, 6.25; Gd, 21.19; N, 9.44.

Fnd.: C, 43.90; H, 6.40; Gd, 20.80; N, 9.33.

Example 14 a)10-[4-(Benzyloxycarbonyl)-2-oxo-3-azabutyl]-1,4,7-α,α′,α″-tris(cyclohexyl)-1,4,7-tris-(benzyloxycarbonylmethyl)-1,4,7,10-tetraazacyclododecane

18.9 g (50 mmol) of1-[4-(benzyloxycarbonyl)-2-oxo-3-azabutyl]-1,4,7,10-tetraazacyclododecanethat is described in Example 13a as an intermediate product and 60 ml(0.35 mol) of N-ethyldiisopropylamine in 200 ml of dichloromethane areadded to 76.1 g (0.2 mol) of2-(trifluoromethanesulfonyloxy)-2-cyclohexylacetic acid benzyl ester(Qabar et al., Tetrahedron Letters (1998), 39(33), 5895) in 400 ml ofdichloromethane, and it is stirred for 6 hours under reflux and thenovernight at room temperature. It is extracted three times with 500 mlof water each, the organic phase is dried on magnesium sulfate andevaporated to the dry state. The residue is chromatographed on silicagel (mobile solvent: dichloromethane/methanol: 20/1). The fractions thatcontain the product are combined and concentrated by evaporation.

Yield: 38.5 g (72% of theory) of a colorless, crystalline powder

Elementary analysis:

Cld.: C, 71.95; H, 8.02; N, 6.56.

Fnd.: C, 71.90; H, 8.21; N, 6.73.

b)10-(4-Carboxy-2-oxo-3-azabutyl)-1,4,7-α,α′,α″-tris(cyclohexyl)-1,4,7-tris(carboxymethyl)-1,4,7,10-tetraazacyclododecane

32.1 g (30 mmol) of the title compound of Example 14a is dissolved in400 ml of isopropanol, mixed with 40 ml of water, and 3 g of palladiumcatalyst (10% Pd/C) is added. It is hydrogenated for 8 hours at 50° C.Catalyst is filtered out, and the filtrate is evaporated to the drystate in a vacuum.

Yield: 21.2 g (quantitative) of a colorless powder

Elementary analysis:

Cld.: C, 61.08; H, 8.69; N, 9.89.

Fnd.: C, 61.27; H, 8.55; N, 9.41.

c) Gd Complex of10-(4-carboxy-2-oxo-3-azabutyl)-1,4,7-α,α′,α″-tris(cyclohexyl)-1,4,7-tris(carboxymethyl)-1,4,7,10-tetraazacyclododecane

14.2 g (20 mmol) of the ligand that is described in Example 14b isdissolved in 150 ml of water and 150 ml of isopropanol, and it isacidified by adding 5 ml of acetic acid. 3.6 g (10 mmol) of gadoliniumoxide is added, and it is refluxed for 8 hours. After complexing iscompleted, it is set at pH 7.4 again with ammonia and chromatographed onsilica gel (mobile solvent: dichloromethane/methanol/ammonia: 20/20/1).The fractions that contain the product are combined and evaporated tothe dry state. The residue is taken up with formic acid and evaporatedto the dry state several times with the addition of dichloromethane andthen dried in a vacuum until a constant weight is reached.

Yield: 13.5 g (71% of theory) of a colorless powder.

Water content (Karl-Fischer): 9.0%

Elementary analysis (relative to anhydrous substance):

Cld.: C, 50.15; H, 6.78; Gd, 18.24; N, 8.12.

Fnd.: C, 49.92; H, 6.51; Gd, 18.01; N, 8.31.

Example 15 a)10-[4-(Benzyloxycarbonyl)-1-methyl-2-oxo-3-azabutyl]-2,5,8,11-tetramethyl-1,4,7,10-tetraazacyclododecane-1,4,7-triaceticacid-tri-t-butylester, sodium bromide complex

0.50 g (1.67 mmol) of 2-bromo-propionylglycine-benzyl ester (Example 1eof WO 98/24774) is added to 1.14 g (5 mmol) of2,5,8,11-tetramethyl-1,4,7,10-tetraazacyclododecane (Petrov et al., DE19608307; Ranganathan et al., WO 95/31444), dissolved in 10 ml ofchloroform, and it is stirred overnight at room temperature. 250 ml ofwater is added, the organic phase is separated, and it is washed twicein each case with 200 ml of water. The organic phase is dried onmagnesium sulfate and evaporated to the dry state in a vacuum. Theresidue is chromatographed on silica gel (mobile solvent:chloroform/methanol/aqueous 25% ammonia=10/5/1). 822 mg (4.2 mmol) ofbromoacetic acid-tert-butyl ester is added to the thus obtained1-[4-(benzyloxycarbonyl)-1-methyl-2-oxo-3-azabutyl]-2,5,8,11-tetramethyl-1,4,7,10-tetraazacyclododecane(0.70 g; 1.27 mmol; 76% of theory) and 541 mg (5.1 mmol) of sodiumcarbonate in 5 ml of acetonitrile, and it is stirred for 12 hours at 60°C. It is cooled to 0° C., and salts are filtered out. The filtrate isevaporated to the dry state, and the residue is chromatographed onsilica gel (mobile solvent: methylene chloride/methanol=20:1).

Yield: 964 mg (85% of theory) of a colorless solid

Elementary analysis:

Cld.: C, 56.49; H, 8.01; N, 7.84; Na, 2.57; Br, 8.95.

Fnd.: C, 56.37; H, 7.88; N, 7.61; Na, 2.33; Br, 8.59.

b)10-(4-Carboxy-1-methyl-2-oxo-3-azabutyl)-2,5,8,11-tetramethyl-1,4,7,10-tetraazacyclododecane-1,4,7-triaceticacid-tri-tert-butyl ester (sodium bromide complex)

893 mg (1.0 mmol) of the title compound of Example 15a is dissolved in10 ml of isopropanol, and a spatula tip full of palladium catalyst (10%Pd/C) is added. It is hydrogenated overnight at room temperature.Catalyst is filtered out, and the filtrate is evaporated to the drystate. The residue is recrystallized from dioxane.

Yield: 562 mg (70% of theory) of a crystalline solid

Elementary analysis:

Cld.: C, 52.36; H, 8.16; N, 8.72; Na, 2.86; Br, 9.95.

Fnd.: C, 52.51; H, 8.30; N, 8.93; Na, 2.71; Br, 9.44.

c) Gadolinium complex of10-(4-carboxy-1-methyl-2-oxo-3-azabutyl)-2,5,8,11-tetramethyl-1,4,7,10-tetraazacyclododecane-1,4,7-triaceticacid

803 mg (1.0 mmol) of the title compound of Example 15b is dissolved in 5ml of trifluoroacetic acid and stirred for 3 hours at room temperature.It is evaporated to the dry state, the residue is taken up in 300 ml ofwater, and the solution is added to a column, filled with Reillex® 425PVP. It is eluted with water. The product-containing fractions arecombined and evaporated to the dry state (446 mg; 0.84 mmol) and againdissolved in 4 ml of water. 152 mg (0.42 mmol) of gadolinium oxide isadded, and it is heated for 3 hours to 90° C. It is evaporated to thedry state (vacuum), and the residue is crystallized from 90% aqueousethanol. The crystals are suctioned off, washed once with ethanol, thenwith acetone and finally with dimethyl ether and dried in a vacuumfurnace at 130° C. (24 hours).

Yield: 469 mg (65% of theory) of a colorless, crystalline powder

Water content: 5%

Elementary analysis (relative to anhydrous substance):

Cld.: C, 40.28; H, 5.58; N, 10.21; Gd, 22.93.

Fnd.: C, 40.06; H, 5.75; N, 10.43; Gd, 22.40.

Example 16 Gd Complex of10-[8-(N-maleimido)-1-methyl-2,5-dioxo-3,6-diazaoctyl]-1,4,7-α,α′,α″-tris-(isopropyl)-1,4,7-tris(carboxymethyl)-1,4,7,10-tetraazacyclododecane

2.27 g (3 mmol) of the Gd complex acid that is described in Example 2 isdissolved in 15 ml of DMF, mixed with 380 mg (3.3 mmol) ofN-hydroxysuccinimide and 681 mg (3.3 mmol) of dicyclohexylcarbodiimidewhile being cooled with ice and preactivated for 1 hour in ice. Then, amixture that consists of 839 mg (3.3 mmol) of N-(2-aminoethyl)maleimidetrifluoroacetate salt (Arano et al., J. Med. Chem., 1996, 39, 3458) and0.7 ml (4 mmol) of N,N-diisopropylethyl-amine in 10 ml of DMF is addedand stirred overnight at room temperature. The reaction mixture iscooled again in an ice bath, filtered, and the filtrate is evaporated tothe dry state in a vacuum. The residue is chromatographed on silica gel(mobile solvent: dichloromethane/methanol: 1/1).

Yield: 997 mg (35% of theory)

Water content (Karl-Fischer): 7.5%

Elementary analysis (relative to anhydrous substance):

Cld.: C, 46.51; H, 6.20; Gd, 17.91; N, 11.17.

Fnd.: C, 46.28; H, 6.44; Gd, 17.31; N, 11.26.

Example 17 Gd Complex of10-[8-(N-maleimido)-1-methyl-2,5-dioxo-3,6-diazaoctyl]-1,4,7-α,α′,α″-tris-(cyclohexyl)-1,4,7-tris(carboxymethyl)-1,4,7,10-tetraazacyclododecane

2.63 g (3 mmol) of the Gd complex acid that is described in Example 3 isdissolved in 15 ml of DMF, mixed with 380 mg (3.3 mmol) ofN-hydroxysuccinimide and 681 mg (3.3 mmol) of dicyclohexylcarbodiimidewhile being cooled with ice, and preactivated for 1 hour in ice. Then, amixture that consists of 839 mg (3.3 mmol) of N-(2-aminoethyl)maleimidetrifluoroacetate salt (Arano et al., J. Med. Chem., 1996, 39, 3458) and0.7 ml (4 mmol) of N,N-diisopropylethylamine in 10 ml of DMF is addedand stirred overnight at room temperature. The reaction mixture iscooled again in an ice bath, filtered, and the filtrate is evaporated tothe dry state in a vacuum. The residue is chromatographed on silica gel(mobile solvent: dichloromethane/methanol: 1/1).

Yield: 1.24 g (39% of theory)

Water content (Karl-Fischer): 6.0%

Elementary analysis (relative to anhydrous substance):

Cld.: C, 51.74; H, 6.66; Gd, 15.75; N, 9.82.

Fnd.: C, 51.77; H, 6.41; Gd, 15.25; N, 10.02.

Example 18 a) (3-Bromo-2-oxo-pyrrolidin-1-yl)acetic acid benzyl ester

67.7 g (0.2 mol) of glycinebenzyl ester tosylate and 61.2 ml (0.44 mol)of triethylamine are dissolved in 200 ml of methylene chloride and addedin drops at 0° C. to a solution of 52.9 g (0.2 mol) of2,4-dibromobutyric acid chloride (Gramain et al. Synth. Commun. (1997),(27), 1827) in 200 ml of methylene chloride within 45 minutes, and it isstirred for 18 hours at room temperature. The reaction mixture is nowadded in drops at 0° C. to a solution of 400 ml of aqueous 32% sodiumhydroxide and 2 g of tetrabutylammonium hydrogen carbonate (about 15minutes), and it is stirred for 30 minutes. Then, the phases areseparated, and the aqueous phase is extracted three times with 200 mleach of dichloromethane. The organic phases are dried on sodium sulfate,the solution is evaporated to the dry state and chromatographed onsilica gel (methylene chloride). The fractions that contain the productare combined and concentrated by evaporation.

Yield: 29.3 g (47% of theory)

Elementary analysis:

Cld.: C, 50.02; H, 4.52; N, 4.49.

Fnd.: C, 50.34; H, 4.44; N, 4.41.

b)10-[1-(Benzyloxycarbonylmethyl)-2-oxo-pyrrolidin-3-yl]-1,4,7-α,α′,α″-trimethyl-1,4,7-tris-(benzyloxycarbonylmethyl)-1,4,7,10-tetraazacyclododecane

20.7 (66.3 mmol) of (3-bromo-2-oxo-pyrrolidin-1-yl)acetic acid benzylester is added to 28.7 g (165.8 mmol) of 1,4,7,10-tetraazacyclododecane,dissolved in 300 ml of chloroform, and it is stirred overnight at roomtemperature. 250 ml of water is added, the organic phase is separated,and it is washed twice in each case with 200 ml of water. The organicphase is dried on magnesium sulfate and evaporated to the dry state in avacuum. The residue is chromatographed on silica gel (mobile solvent:chloroform/methanol/aqueous 25% ammonia=10/5/1). The thus obtained1-[1-(benzyloxycarbonylmethyl)-2-oxo-pyrrolidin-3-yl]-1,4,7,10-tetraazacyclododecane(20.9 g; 51.8 mmol; 78% of theory) and 60 ml (0.35 mol) ofN-ethyldiisopropylamine in 200 ml of dichloromethane are added to 62.45g (0.2 mol) of 2-(trifluoromethanesulfonyloxy)propanoic acid benzylester (Kitazaki et al., Chem. Pharm. Bull. (1999), 47(3), 360) in 400 mlof dichloromethane, and it is stirred for 6 hours under reflux and thenovernight at room temperature. It is extracted three times with 500 mlof water each, the organic phase is dried on magnesium sulfate andevaporated to the dry state. The residue is chromatographed on silicagel (mobile solvent: dichloromethane/methanol: 20/1). The fractions thatcontain the product are combined and concentrated by evaporation.

Yield: 32.7 g (71% of theory) of a colorless, crystalline powder

Elementary analysis:

Clod.: C, 68.82; H, 7.13; N, 7.87.

Find.: C, 68.54; H, 7.28; N, 8.01.

c)10-[1-(Carboxymethyl)-2-oxo-pyrrolidin-3-yl]-1,4,7-α,α′,α″-trimethyl-1,4,7-tris(carboxymethyl)-1,4,7,10-tetraazacyclododecane

26.7 g (30 mmol) of the title compound of Example 18b is dissolved in400 ml of isopropanol, mixed with 40 ml of water, and 3 g of palladiumcatalyst (10% Pd/C) is added. It is hydrogenated for 8 hours at 50° C.Catalyst is filtered out, and the filtrate is evaporated to the drystate in a vacuum.

Yield: 15.8 g (quantitative) of a colorless powder

Elementary analysis:

Cld.: C, 52.16; H, 7.42; N, 13.22.

Fnd.: C, 52.32; H, 7.35; N, 13.11.

d) Gd Complex of10-[1-(carboxymethyl)-2-oxo-pyrrolidin-3-yl]-1,4,7-α,α′,α″-trimethyl-1,4,7-tris(carboxymethyl)-1,4,7,10-tetraazacyclododecane

10.6 g (20 mmol) of the ligand that is described in Example 18c isdissolved in 200 ml of water and 80 ml of isopropanol and acidified byadding 5 ml of acetic acid. 3.6 g (10 mmol) of gadolinium oxide isadded, and it is refluxed for 3 hours. After complexing is completed, itis set at pH 7.4 again with ammonia and chromatographed on silica gel(mobile solvent: dichloromethane/methanol/ammonia: 20/20/1). Thefractions that contain the product are combined and added via an IR-120®cation exchange column (H⁺ form). The acidic elate is freeze-dried.

Yield: 9.7 g (67% of theory) of a colorless powder.

Water content (Karl-Fischer): 8.3%

Elementary analysis (relative to anhydrous substance)

Cld.: C, 40.40; H, 5.31; Gd, 23.00; N, 10.24.

Fnd.: C, 39.99; H, 5.55; Gd, 22.93; N, 10.45.

Example 19 a)10-[1-(Benzyloxycarbonylmethyl)-2-oxo-pyrrolidin-3-yl]-1,4,7-α,α′,α″-tris(isopropyl)-1,4,7-tris(benzyloxycarbonylmethyl)-1,4,7,10-tetraazacyclododecane

20.2 g (50 mmol) of1-[1-(benzyloxycarbonylmethyl)-2-oxo-pyrolidin-3-yl]-1,4,7,10-tetraazacyclododecanethat is described in Example 18b as an intermediate product and 60 ml(0.35 mmol) of N-ethyldiisopropylamine in 200 ml of dichloromethane areadded to 68.1 g (0.2 mol) of 2-(trifluoromethanesulfonyloxy)-isovalericacid benzyl ester (Walker et al., Tetrahedron (1997), 53(43), 14591) in400 ml of dichloromethane, and it is stirred for 6 hours under refluxand then overnight at room temperature. It is extracted three times with500 ml of water each, the organic phase is dried on magnesium sulfateand evaporated to the dry state. The residue is chromatographed onsilica gel (mobile solvent: dichloromethane/methanol: 20/1). Thefractions that contain the product are combined and concentrated byevaporation.

Yield: 34.1 g (70% of theory) of a colorless, crystalline powder

Elementary analysis:

Cld.: C, 70.27; H, 7.76; N, 7.19.

Fnd.: C, 70.45; H, 7.61; N, 7.11.

b)10-[1-(Carboxymethyl)-2-oxo-pyrrolidin-3-yl]-1,4,7-α,α′,α″-tris(isopropyl)-1,4,7-tris(carboxymethyl)-1,4,7,10-tetraazacyclododecane

29.2 g (30 mmol) of the title compound of Example 19a is dissolved in400 ml of isopropanol, mixed with 40 ml of water, and 3 g of palladiumcatalyst (10% Pd/C) is added. It is hydrogenated for 8 hours at 50° C.Catalyst is filtered out, and the filtrate is evaporated to the drystate in a vacuum.

Yield: 18.4 g (quantitative) of a colorless powder

Elementary analysis:

Cld.: C, 56.75; H, 8.38; N, 11.41.

Fnd.: C, 56.89; H, 8.31; N, 11.37.

c) Gd Complex of10-[1-(carboxymethyl)-2-oxo-pyrrolidin-3-yl]-1,4,7-α,α′,α″-tris(isopropyl)-1,4,7-tris(carboxymethyl)-1,4,7,10-tetraazacyclododecane

12.3 g (20 mmol) of the ligand that is described in Example 19b isdissolved in 200 ml of water and 80 ml of isopropanol and acidified byadding 5 ml of acetic acid. 3.6 g (10 mmol) of gadolinium oxide isadded, and it is refluxed for 3 hours. After complexing is completed, itis set at pH 7.4 with ammonia and chromatographed on silica gel (mobilesolvent: dichloromethane/methanol/ammonia: 20/20/1). The fractions thatcontain the product are combined and added via an IR-120® cationexchange column (H⁺ form). The acidic eluate is freeze-dried.

Yield: 11.9 g (75% of theory) of a colorless powder.

Water content (Karl-Fischer): 8.2%

Elementary analysis (relative to anhydrous substance):

Cld.: C, 45.36; H, 6.30; Gd, 20.48; N, 9.12.

Fnd.: C, 45.89; H, 6.22; Gd, 20.23; N, 9.01.

The Dy complex of10-[1-(carboxymethyl)-2-oxo-pyrrolidin-3-yl]-1,4,7-α,α′,α″-tris(isopropyl)-1,4,7-tris(carboxymethyl)-1,4,7,10-tetraazacyclododecaneis obtained analogously with use of 12.3 g (20 mmol) of the ligand thatis described in Example 19b and 3.73 g (10 mmol) of dysprosium oxideinstead of gadolinium oxide.

Yield: 11.4 g (71% of theory) of a colorless powder.

Water content (Karl-Fischer): 8.0%

Elementary analysis (relative to anhydrous substance):

Cld.: C, 45.05; H, 6.26; Dy, 21.02; N, 9.06.

Fnd.: C, 45.35; H, 6.22; Dy, 20.88; N, 9.04.

Example 20 a)10-[1-(Benzyloxycarbonylmethyl)-2-oxo-pyrrolidin-3-yl]-1,4,7-α,α′,α″-tris(cyclohexyl)-1,4,7-tris(benzyloxycarbonylmethyl)-1,4,7,10-tetraazacyclododecane

20.2 g (50 mmol) of1-[1-(benzyloxycarbonylmethyl)-2-oxo-pyrrolidin-3-yl]-1,4,7,10-tetraazacyclododecanethat is described in Example 18b as an intermediate product and 60 ml(0.35 mol) of N-ethyldiisopropylamine in 200 ml of dichloromethane areadded to 76.1 g (0.2 mol) of2-(trifluoromethanesulfonyloxy)-2-cyclohexylacetic acid benzyl ester(Qabar et al., Tetrahedron Letters (1998), 39(33), 5895) in 400 ml ofdichloromethane, and it is stirred for 6 hours under reflux and thenovernight at room temperature. It is extracted three times with 500 mlof water each, the organic phase is dried on magnesium sulfate andevaporated to the dry state. The residue is chromatographed on silicagel (mobile solvent: dichloromethane/methanol: 20/1). The fractions thatcontain the product are combined and concentrated by evaporation.

Yield: 37.2 g (68% of theory) of a colorless, crystalline powder

Elementary analysis:

Cld.: C, 72.43; H, 8.01; N, 6.40.

Fnd.: C, 72.55; H, 7.98; N, 6.35.

b)10-[1-(Carboxymethyl)-2-oxo-pyrrolidin-3-yl]-1,4,7-α,α′,α″-tris(cyclohexyl)-1,4,7-tris(carboxymethyl)-1,4,7,10-tetraazacyclododecane

32.8 g (30 mmol) of the title compound of Example 20a is dissolved in400 ml of isopropanol, mixed with 40 ml of water, and 3 g of palladiumcatalyst (10% Pd/C) is added. It is hydrogenated for 8 hours at 50° C.Catalyst is filtered out, and the filtrate is evaporated to the drystate in a vacuum.

Yield: 22.0 g (quantitative) of a colorless powder

Elementary analysis:

Cld.: C, 62.19; H, 8.65; N, 9.54.

Fnd.: C, 62.44; H, 8.56; N, 9.46.

c) Gd Complex of10-[1-(carboxymethyl)-2-oxo-pyrrolidin-3-yl]-1,4,7-α,α′,α″-tris(cyclohexyl)-1,4,7-tris(carboxymethyl)-1,4,7,10-tetraazacyclododecane

14.6 g (20 mmol) of the ligand that is described in Example 20b isdissolved in 150 ml of water and 150 ml of isopropanol and acidified byadding 5 ml of acetic acid. 3.6 g (10 mmol) of gadolinium oxide isadded, and it is refluxed for 8 hours. After complexing is completed, itis set at pH 7.4 again with ammonia and chromatographed on silica gel(mobile solvent: dichloromethane/methanol/ammonia: 20/20/1). Thefractions that contain the product are combined and evaporated to thedry state. The residue is taken up with formic acid and evaporated tothe dry state several times with the addition of dichloromethane, andthen it is dried in a vacuum until a constant weight is reached.

Yield: 12.1 g (65% of theory) of a colorless powder.

Water content (Karl-Fischer): 7.0%

Elementary analysis (relative to anhydrous substance):

Cld.: C, 51.39; H, 6.81; Gd, 17.70; N, 7.89.

Fnd.: C, 51.64; H, 6.77; Gd, 17.44; N, 7.77.

Example 21 a) (3-Bromo-2-oxo-pyrrolidin-1-yl)benzoic acid benzyl ester

45.5 g (0.2 mol) of 4-aminobeznoic acid benzyl ester and 30.6 ml (0.22mol) of triethylamine are dissolved in 200 ml of methylene chloride andadded in drops at 0° C. to a solution of 52.9 g (0.2 mol) of2,4-dibromobutyric acid chloride (Gramin et al. Synth. Commun. (1997),(27), 1827) in 200 ml of methylene chloride within 45 minutes, and it isstirred for 18 hours at room temperature. The reaction mixture is nowadded in drops at 0° C. to a solution of 400 ml of aqueous 32% sodiumhydroxide and 2 g of tetrabutylammonium hydrogen carbonate (about 15minutes), and it is stirred for 30 minutes. Then, the phases areseparated, and the aqueous phase is extracted three times with 200 ml ofdichloromethane each. The organic phases are dried on sodium sulfate,the solution is evaporated to the dry state and chromatographed onsilica gel (methylene chloride). The fractions that contain the productare combined and concentrated by evaporation.

Yield: 38.2 g (51% of theory)

Elementary analysis:

Cld.: C, 57.77; H, 4.31; N, 3.74.

Fnd.: C, 57.99; H, 4.27; N, 3.66.

b)10-[1-(4-Benzyloxycarbonylphenyl)-2-oxo-pyrrolidin-3-yl]-1,4,7-α,α′,α″-trimethyl-1,4,7-tris-(benzyloxycarbonylmethyl)-1,4,7,10-tetraazacyclododecane

26.9 g (71.9 mmol) of (3-bromo-2-oxo-pyrrolidin-1-yl)benzoic acid benzylester is added to 31.2 g (180 mmol) of 1,4,7,10-tetraazacyclododecane,dissolved in 300 ml of chloroform, and it is stirred overnight at roomtemperature. 250 ml of water is added, the organic phase is separated,and it is washed twice in each case with 200 ml of water. The organicphase is dried on magnesium sulfate and evaporated to the dry state in avacuum. The residue is chromatographed on silica gel (mobile solvent:chloroform/methanol/aqueous 25% ammonia=10/5/1). The thus obtained1-[1-(4-benzyloxycarbonylphenyl)-2-oxo-pyrrolidin-3-yl]-1,4,7,10-tetraazacyclododecane(26.1 g; 56.1 mmol; 78% of theory) and 60 ml (0.35 mol) ofN-ethyldiisopropylamine in 200 ml of dichloromethane are added to 62.45g (0.2 mol) of 2-(trifluoromethanesulfonyloxy)propanoic acid benzylester (Kitazaki et al., Chem. Pharm. Bull. (1999), 47(3), 360) in 400 mlof dichloromethane, and it is stirred for 6 hours under reflux and thenovernight at room temperature. It is extracted three times with 500 mlof water each, the organic phase is dried on magnesium sulfate andevaporated to the dry state. The residue is chromatographed on silicagel (mobile solvent: dichloromethane/methanol: 20/1). The fractions thatcontain the product are combined and concentrated by evaporation.

Yield: 36.3 g (68% of theory) of a colorless, crystalline powder

Elementary analysis:

Cld.: C, 70.64; H, 6.88; N, 7.36.

Fnd.: C, 70.89; H, 6.81; N, 7.29.

c)10-[1-(4-Carboxyphenyl)-2-oxo-pyrrolidin-3-y]-1,4,7-α,α′,α″-trimethyl-1,4,7-tris(carboxymethyl)-1,4,7,10-tetraazacyclododecane

28.6 g (30 mmol) of the title compound of Example 21 b is dissolved in400 ml of isopropanol, mixed with 40 ml of water, and 3 g of palladiumcatalyst (10% Pd/C) is added. It is hydrogenated for 8 hours at 50° C.Catalyst is filtered out, and the filtrate is evaporated to the drystate in a vacuum.

Yield: 17.7 g (quantitative) of a colorless powder

Elementary analysis:

Cld.: C, 56.84; H, 6.98; N, 11.84.

Fnd.: C, 57.04; H, 6.91; N, 11.79.

d) Gd Complex of10-[1-(4-carboxyphenyl)-2-oxo-pyrrolidin-3-yl]-1,4,7-α,α′,α″-trimethyl-1,4,7-tris(carboxymethyl)-1,4,7,10-tetraazacyclododecane

11.8 g (20 mmol) of the ligand that is described in Example 21c isdissolved in 200 ml of water and 80 ml of isopropanol, and it isacidified by adding 5 ml of acetic acid. 3.6 g (10 mmol) of gadoliniumoxide is added, and it is refluxed for 3 hours. After complexing iscompleted, it is set at pH 7.4 again with ammonia and chromatographed onsilica gel (mobile solvent: dichloromethane/methanol/ammonia: 20/20/1).The fractions that contain the product are combined and added via anIR-120® cation exchange column (H⁺ form). The acidic eluate isfreeze-dried.

Yield: 11.1 g (71% of theory) of a colorless powder.

Water content (Karl-Fischer): 7.5%

Elementary analysis (relative to anhydrous substance):

Cld.: C, 45.09; H, 5.13; Gd, 21.08; N, 9.39.

Fnd.: C, 45.45; H, 5.11; Gd, 20.78; N, 9.40.

Example 22 a)10-[1-(4-Benzyloxycarbonylphenyl)-2-oxo-pyrrolidin-3-yl]-1,4,7-α,α′,α″-tris(isopropyl)-1,4,7-tris(benzyloxycarbonylmethyl)-1,4,7,10-tetraazacyclododecane

23.3 g (50 mmol) of1-[1-(4-benzyloxycarbonylphenyl)-2-oxo-pyrrolidin-3-yl]-1,4,7,10-tetraazacyclododecanethat is described in Example 21b as an intermediate product and 60 ml(0.35 mol) of N-ethyldiisopropylamine in 200 ml of dichloromethane areadded to 68.1 g (0.2 mol) of 2-(trifluoromethanesulfonyloxy)-isovalericacid benzyl ester (Walker et al., Tetrahedron (1997), 53(43), 14591) in400 ml of dichloromethane, and it is stirred for 6 hours under refluxand then overnight at room temperature. It is extracted three times with500 ml of water each, the organic phase is dried on magnesium sulfateand evaporated to the dry state. The residue is chromatographed onsilica gel (mobile solvent: dichloromethane/methanol: 20/1). Thefractions that contain the product are combined and concentrated byevaporation.

Yield: 35.3 g (68% of theory) of a colorless, crystalline powder

Elementary analysis:

Cld.: C, 71.86; H, 7.49; N, 6.76.

Fnd.: C, 71.99; H, 7.46; N, 6.71.

b)10-[1-(4-Carboxyphenyl)-2-oxo-pyrrolidin-3-yl]-1,4,7-α,α′,α″-tris(isopropyl)-1,4,7-tris(carboxymethyl)-1,4,7,10-tetraazacyclododecane

31.1 g (30 mmol) of the title compound of Example 22a is dissolved in400 ml of isopropanol, mixed with 40 ml of water, and 3 g of palladiumcatalyst (10% Pd/C) is added. It is hydrogenated for 8 hours at 50° C.Catalyst is filtered out, and the filtrate is evaporated to the drystate in a vacuum.

Yield: 20.2 g (quantitative) of a colorless powder

Elementary analysis:

Cld.: C, 60.43; H, 7.90; N, 10.36.

Fnd.: C, 60.59; H, 7.82; N, 10.31.

c) Gd Complex of10-[1-(4-carboxyphenyl)-2-oxo-pyrrolidin-3-yl]-1,4,7-α,α′,α″-tris(isopropyl)-1,4,7-tris(carboxymethyl)-1,4,7,10-tetraazacyclododecane

13.5 g (20 mmol) of the ligand that is described in Example 22b isdissolved in 200 ml of water and 80 ml of isopropanol, and it isacidified by adding 5 ml of acetic acid. 3.6 g (10 mmol) of gadoliniumoxide is added, and it is refluxed for 3 hours. After complexing iscompleted, it is set at pH 7.4 with ammonia and chromatographed onsilica gel (mobile solvent: dichloromethane/methanol/ammonia: 20/20/1).The fractions that contain the product are combined and added via anIR-120® cation exchange column (H⁺ form). The acidic eluate isfreeze-dried.

Yield: 12.4 g (72% of theory) of a colorless powder.

Water content (Karl-Fischer): 7.8%

Elementary analysis (relative to anhydrous substance):

Cld.: C, 49.20; H, 6.07; Gd, 18.94; N, 8.44.

Fnd.: C, 49.51; H, 6.04; Gd, 18.71; N, 8.45.

The Dy complex of10-[1-(4-carboxyphenyl)-2-oxo-pyrrolidin-3-yl]-1,4,7-α,α′,α″-tris(isopropyl)-1,4,7-tris(carboxymethyl)-1,4,7,10-tetraazacyclododecaneis analogously obtained with use of 13.5 g (20 mmol) of the ligand thatis described in Example 22b and 3.73 g (10 mmol) of dysprosium oxideinstead of gadolinium oxide.

Yield: 13.0 g (75% of theory) of a colorless powder.

Water content (Karl-Fischer): 7.5%

Elementary analysis (relative to anhydrous substance):

Cld.: C, 48.89; H, 6.03; Dy, 19.45; N, 8.38.

Fnd.: C, 49.11; H, 6.04; Dy, 19.22; N, 8.36.

Example 23 a)10-[1-(4-Benzyloxycarbonylphenyl)-2-oxo-pyrrolidin-3-yl]-1,4,7-α,α′,α″-tris(cyclohexyl)-1,4,7-tris(benzyloxycarbonylmethyl)-1,4,7,10-tetraazacyclododecane

23.3 g (50 mmol) of1-[1-(4-benzyloxycarbonylphenyl)-2-oxo-pyrrolidin-3-yl]-1,4,7,10-tetraazacyclododecanethat is described in Example 21b as an intermediate product and 60 ml(0.35 mol) of N-ethylodiisopropylamine in 200 ml of dichloromethane areadded to 76.1 g (0.2 mol) of2-(trifluoromethanesulfonyloxy)-2-cyclohexyalacetic acid benzyl ester(Qabar et al., Tetrahedron Letters (1998), 39(33), 5895) in 400 ml ofdichloromethane, and it is stirred for 6 hours under reflux and thenovernight at room temperature. It is extracted three times with 500 mlof water each, the organic phase is dried on magnesium sulfate andevaporated to the dry state. The residue is chromatographed on silicagel (mobile solvent: dichloromethane/methanol: 20/1). The fractions thatcontain the product are combined and concentrated by evaporation.

Yield: 41.1 g (71% of theory) of a colorless, crystalline powder

Elementary analysis:

Cld.: C, 73.74; H, 7.76; N, 6.06.

Fnd.: C, 73.91; H, 7.69; N, 6.01.

b)10-[1-(4-Carboxyphenyl)-2-oxo-pyrrolidin-3-yl]-1,4,7-α,α′,α″-tris(cyclohexyl)-1,4,7-tris-(carboxymethyl)-1,4,7,10-tetraazacyclododecane

34.7 g (30 mmol) of the title compound of Example 23a is dissolved in400 ml of isopropanol, mixed with 40 ml of water, and 3 g of palladiumcatalyst (10% Pd/C) is added. It is hydrogenated for 8 hours at 50° C.Catalyst is filtered out, and the filtrate is evaporated to the drystate in a vacuum.

Yield: 23.8 g (quantitative) of a colorless powder

Elementary analysis:

Cld.: C, 64.88; H, 8.23; N, 8.80.

Fnd.: C, 65.04; H, 8.19; N, 8.70.

c) Gd Complex of10-[1-(4-carboxyphenyl)-2-oxo-pyrrolidin-3-yl]-1,4,7-α,α′,α″-tris(cyclohexyl)-1,4,7-tris(carboxymethyl)-1,4,7,10-tetraazacyclododecane

15.9 g (20 mmol) of the ligand that is described in Example 23b isdissolved in 150 ml of water and 150 ml of isopropanol and acidified byadding 5 ml of acetic acid.

3.6 g (10 mmol) of gadolinium oxide is added, and it is refluxed for 8hours. After complexing is completed, it is set at pH 7.4 again withammonia and chromatographed on silica gel (mobile solvent:dichloromethane/methanol/ammonia: 20/20/1). The fractions that containthe product are combined and evaporated to the dry state. The residue istaken up with formic acid and evaporated to the dry state several timeswith the addition of dichloromethane and then dried in a vacuum until aconstant weight is reached.

Yield: 12.9 g (65% of theory) of a colorless powder.

Water content (Karl-Fischer): 7.0%

Elementary analysis (relative to anhydrous substance):

Cld.: C, 54.35; H, 6.58; Gd, 16.55; N, 7.37.

Fnd.: C, 54.66; H, 6.57; Gd, 16.32; N, 7.32.

Example 24 a) (3-Bromo-2-oxo-piperidin-1-yl)acetic acid benzyl ester

67.7 g (0.2 mol) of glycine benzyl ester tosylate and 61.2 ml (0.44 mol)of triethylamine are dissolved in 200 ml of methylene chloride and addedin drops at 0° C. to a solution of 55.7 g (0.2 mol) of2,5-dibromovaleric acid chloride (Okawara et al. Chem. Pharm. Bull.(1982), (30), 1225) in 200 ml of methylene chloride within 45 minutes,and it is stirred for 18 hours at room temperature. The reaction mixtureis now added in drops at 0° C. to a solution of 400 ml of aqueous 32%sodium hydroxide and 2 g of tetrabutylammonium hydrogen carbonate (about15 minutes), and it is stirred for 30 minutes. Then, the phases areseparated, and the aqueous phase is extracted three times with 200 ml ofdichloromethane each. The organic phases are dried on sodium sulfate,the solution is evaporated to the dry state and chromatographed onsilica gel (methylene chloride). The fractions that contain the productare combined and concentrated by evaporation.

Yield: 33.2 g (51% of theory)

Elementary analysis:

Cld.: C, 51.55; H, 4.94; N, 4.29.

Fnd.: C, 51.86; H, 4.91; N, 4.18

b)10-[1-(Benzyloxycarbonylmethyl)-2-oxo-piperidin-3-yl]-1,4,7-α,α′,α″-trimethyl-1,4,7-tris-(benzyloxycarbonylmethyl)-1,4,7,10-tetraazacyclododecane

18.9 g (58 mmol) of (3-bromo-2-oxo-piperidin-1-yl)acetic acid benzylester is added to 30.3 g (175 mmol) of 1,4,7,10-tetraazacyclododecane,dissolved in 300 ml of chloroform, and it is stirred overnight at roomtemperature. 250 ml of water is added, the organic phase is separated,and it is washed twice in each case with 200 ml of water. The organicphase is dried on magnesium sulfate and evaporated to the dry state in avacuum. The residue is chromatographed on silica gel (mobile solvent:chloroform/methanol/aqueous 25% ammonia=10/5/1). The thus obtained1-[1-(benzyloxycarbonylmethyl)-2-oxo-piperidin-3-yl]-1,4,7,10-tetraazacyclododecane(20.3 g; 48.6 mol; 84% of theory) and 60 ml (0.35 mol) ofN-ethyldiisopropylamine in 200 ml of dichloromethane are added to 62.45g (0.2 mol) of 2-(trifluoromethanesulfonyloxy)propanoic acid benzylester (Kitazaki et al., Chem. Pharm. Bull. (1999), 47(3), 360) in 400 mlof dichloromethane, and it is stirred for 6 hours under reflux and thenovernight at room temperature. It is extracted three times with 500 mlof water each, and the organic phase is dried on magnesium sulfate andevaporated to the dry state. The residue is chromatographed on silicagel (mobile solvent: dichloromethane/methanol: 20/1). The fractions thatcontain the product are combined and concentrated by evaporation.

Yield: 32.5 g (74% of theory) of a colorless, crystalline powder

Elementary analysis:

Cld.: C, 69.08; H, 7.25; N, 7.75.

Fnd.: C, 69.34; H, 7.19; N, 7.66.

c)10-[1-(Carboxymethyl)-2-oxo-piperidin-3-yl]-1,4,7-α,α′,α″-trimethyl-1,4,7-tris(carboxymethyl)-1,4,7,10-tetraazacyclododecane

27.1 g (30 mmol) of the title compound of Example 24b is dissolved in400 ml of isopropanol, mixed with 40 ml of water, and 3 g of palladiumcatalyst (10% Pd/C) is added. It is hydrogenated for 8 hours at 50° C.Catalyst is filtered out, and the filtrate is evaporated to the drystate in a vacuum.

Yield: 16.3 g (quantitative) of a colorless powder

Elementary analysis:

Cld.: C, 53.03; H, 7.60; N, 12.88.

Fnd.: C, 53.34; H, 7.54; N, 12.79.

d) Gd Complex of10-[1-(Carboxymethyl)-2-oxo-piperidin-3-yl]1,4,7-α,α′,α″-trimethyl-1,4,7-tris(carboxymethyl)-1,4,7,10-tetraazacyclododecane

10.9 g (20 mmol) of the ligand that is described in Example 24c isdissolved in 200 ml of water and 80 ml of isopropanol, and it isacidified by adding 5 ml of acetic acid. 3.6 g (10 mmol) of gadoliniumoxide is added and refluxed for 3 hours. After complexing is completed,it is set at pH 7.4 again with ammonia and chromatographed on silica gel(mobile solvent: dichloromethane/methanol/ammonia: 20/20/1). Thefractions that contain the product are combined and added via an IR-120®cation exchange column (H⁺ form). The acidic eluate is freeze-dried.

Yield: 9.6 g (65% of theory) of a colorless powder.

Water content (Karl-Fischer): 7.2%

Elementary analysis (relative to anhydrous substance):

Cld.: C, 41.31; H, 5.49; Gd, 22.53; N, 10.04.

Fnd.: C, 41.67; H, 5.48; Gd, 22.21; N, 9.97.

Example 25 a)10-[1-(Benzyloxycarbonylmethyl)-2-oxo-piperidin-3-yl]-1,4,7-α,α′,α″-tris(isopropyl)-1,4,7-tris(benzyloxycarbonylmethyl)-1,4,7,10-tetraazacyclododecane

20.9 g (50 mmol) of1-[1-(benzyloxycarbonylmethyl)-2-oxo-piperidin-3-yl]-1,4,7,10-tetraazacyclododecanethat is described in Example 24b as an intermediate product and 60 ml(0.35 mol) of N-ethyldiisopropylamine in 200 ml of dichloromethane areadded to 68.1 g (0.2 mol) of 2-(trifluoromethanesulfonyloxy)-isovalericacid benzyl ester (Walker et al., Tetrahedron (1997), 53(43), 14591) in400 ml of dichloromethane, and it is stirred for 6 hours under refluxand then overnight at room temperature. It is extracted three times with500 ml of water each, and the organic phase is dried on magnesiumsulfate and evaporated to the dry state. The residue is chromatographedon silica gel (mobile solvent: dichloromethane/methanol: 20/1). Thefractions that contain the product are combined and concentrated byevaporation.

Yield: 36.2 g (73% of theory) of a colorless, crystalline powder

Elementary analysis:

Cld.: C, 70.49; H, 7.85; N, 7.09.

Fnd.: C, 70.61; H, 7.83; N, 7.01.

b)10-[1-(Carboxymethyl)-2-oxo-piperidin-3-yl]-1,4,7-α,α′,α″-tris(isopropyl)-1,4,7-tris(carboxymethyl)-1,4,7,10-tetraazacyclododecane

29.6 g (30 mmol) of the title compound of Example 25a is dissolved in400 ml of isopropanol, mixed with 40 ml of water, and 3 g of palladiumcatalyst (10% Pd/C) is added. It is hydrogenated for 8 hours at 50° C.Catalyst is filtered out, and the filtrate is evaporated to the drystate in a vacuum.

Yield: 18.8 g (quantitative) of a colorless powder

Elementary analysis:

Cld.: C, 57.40; H, 8.51; N, 11.16.

Fnd.: C, 57.64; H, 8.45; N, 11.09.

c) Gd Complex of10-[1-(carboxymethyl)-2-oxo-piperidin-3-yl]-1,4,7-α,α′,α″-tris(isopropyl)-1,4,7-tris(carboxymethyl)-1,4,7,10-tetraazacyclododecane

12.6 g (20 mmol) of the ligand that is described in Example 25b isdissolved in 200 ml of water and 80 ml of isopropanol and acidified byadding 5 ml of acetic acid. 3.6 g (10 mmol) of gadolinium oxide isadded, and it is refluxed for 3 hours. After complexing is completed, itis set at pH 7.4 with ammonia and chromatographed on silica gel (mobilesolvent: dichloromethane/methanol/ammonia: 20/20/1). The fractions thatcontain the product are combined and added via an IR-120® cationexchange column (H⁺ form). The acidic eluate is freeze-dried.

Yield: 11.7 g (71% of theory) of a colorless powder.

Water content (Karl-Fischer): 8.1%

Elementary analysis (relative to anhydrous substance):

Cld.: C, 46.08; H, 6.44; Gd, 20.11; N, 8.96.

Fnd.: C, 46.34; H, 6.41; Gd, 19.99; N, 8.91.

The DY complex of10-[1-(carboxymethyl)-2-oxo-piperidin-3-yl]-1,4,7-α,α′,α″-tris(isopropyl)-1,4,7-tris(carboxymethyl)-1,4,7,10-tetraazacyclododecaneis analogously obtained with use of 12.6 g (20 mmol) of the ligand thatis described in Example 25b and 3.73 g (10 mmol) of dysprosium oxideinstead of gadolinium oxide.

Yield: 10.8 g (66% of theory) of a colorless powder.

Water content (Karl-Fischer): 7.6%

Elementary analysis (relative to anhydrous substance):

Cld.: C, 45.77; H, 6.40; Dy, 20.64; N, 8.90.

Fnd.: C, 46.01; H, 6.46; Dy, 20.34; N, 8.91.

Example 26 a)10-[1-(Benzyloxycarbonylmethyl)-2-oxo-piperidin-3-yl]-1,4,7-α,α′,α″-tris(cyclohexyl)-1,4,7-tris(benzyloxycarbonylmethyl)-1,4,7,10-tetraazacyclododecane

20.9 g (50 mmol) of1-[1-(benzyloxycarbonylmethyl)-2-oxo-piperidin-3-yl]-1,4,7,10-tetraazacyclododecanethat is described in Example 24b as an intermediate product and 60 ml(0.35 mol) of N-ethyldiisopropylamine in 200 ml of dichloromethane areadded to 76.1 g (0.2 mol) of2-(trifluoromethanesulfonyloxy)-2-cyclohexylacetic acid benzyl ester(Qabar et al., Tetrahedron Letters (1998), 39(33), 5895) in 400 ml ofdichloromethane, and it is stirred for 6 hours under reflux and thenovernight at room temperature. It is extracted three times with 500 mlof water each, the organic phase is dried on magnesium sulfate andevaporated to the dry state. The residue is chromatographed on silicagel (mobile solvent: dichloromethane/methanol: 20/1). The fractions thatcontain the product are combined and concentrated by evaporation.

Yield: 39.8 g (72% of theory) of a colorless, crystalline powder

Elementary analysis:

Cld.: C, 72.60; H, 8.09; N, 6.32.

Fnd.: C, 72.89; H, 7.98; N, 6.27.

b)10-[1-(Carboxymethyl)-2-oxo-piperidin-3-yl]-1,4,7-α,α′,α″-tris(cyclohexyl)-1,4,7-tris(carboxymethyl)-1,4,7,10-tetraazacyclododecane

33.3 g (30 mmol) of the title compound of Example 26a is dissolved in400 ml of isopropanol, mixed with 40 ml of water, and 3 g of palladiumcatalyst (10% Pd/C) is added. It is hydrogenated for 8 hours at 50° C.Catalyst is filtered out, and the filtrate is evaporated to the drystate in a vacuum.

Yield: 22.4 g (quantitative) of a colorless powder

Elementary analysis:

Cld.: C, 62.63; H, 8.76; N, 9.36.

Fnd.: C, 62.77; H, 8.71; N, 9.29.

c) Gd Complex of10-[1-(carboxymethyl)-2-oxo-piperidin-3-yl]-1,4,7-α,α′,α″-tris(cyclohexyl)-1,4,7-tris(carboxymethyl)-1,4,7,10-tetraazacyclododecane

14.9 g (20 mmol) of the ligand that is described in Example 26b isdissolved in 150 ml of water and 150 ml of isopropanol, and it isacidified by adding 5 ml of acetic acid. 3.6 g (10 mmol) of gadoliniumoxide is added, and it is refluxed for 8 hours. After complexing iscompleted, it is set at pH 7.4 again with ammonia and chromatographed onsilica gel (mobile solvent: dichloromethane/methanol/ammonia: 20/20/1).The fractions that contain the product are combined and evaporated tothe dry state. The residue is taken up with formic acid and evaporatedto the dry state several times with the addition of dichloromethane andthen dried in a vacuum until a constant weight is reached.

Yield: 12.9 g (68% of theory) of a colorless powder.

Water content (Karl-Fischer): 7.6%

Elementary analysis (relative to anhydrous substance):

Cld.: C, 51.92; H, 6.93; Gd 17.43; N, 7.76.

Fnd.: C, 52.09; H, 6.88; Gd, 17.21; N, 7.77.

Example 27 a) (3-Bromo-2-oxo-piperidin-1-yl)benzoic acid benzyl ester

45.5 g (0.2 mol) of 4-aminobenzoic acid benzyl ester and 30.6 ml (0.22mol) of triethylamine are dissolved in 200 ml of methylene chloride andadded in drops within 45 minutes at 0° C. to a solution of 55.3 g (0.2mol) of 2,5-dibromovaleric acid chloride (Okawara et al. Chem. Pharm.Bull. (1982), (30), 1225) in 200 ml of methylene chloride, and it isstirred for 18 hours at room temperature. The reaction mixture is nowadded in drops at 0° C. to a solution of 400 ml of aqueous 32% sodiumhydroxide and 2 g of tetrabutylammonium hydrogen carbonate (about 15minutes), and it is stirred for 30 minutes. Then, the phases areseparated, and the aqueous phase is extracted three times with 200 ml ofdichloromethane each. The organic phases are dried on sodium sulfate,the solution is evaporated to the dry state and chromatographed onsilica gel (methylene chloride). The fractions that contain the productare combined and concentrated by evaporation.

Yield: 38.8 g (50% of theory)

Elementary analysis:

Cld.: C, 58.78; H, 4.67; N, 3.61.

Fnd.: C, 59.01; H, 4.50; N, 3.59.

b)10-[1-(4-Benzyloxycarbonylphenyl)-2-oxo-piperidin-3-yl]-1,4,7-α,α′,α″-trimethyl-1,4,7-tris-(benzyloxycarbonylmethyl)-1,4,7,10-tetraazacyclododecane

26.6 g (68.5 mmol) of (3-bromo-2-oxo-piperidin-1-yl)benzoic acid benzylester is added to 31.2 g (180 mmol) of 1,4,7,10-tetraazacyclododecane,dissolved in 300 ml of chloroform, and it is stirred overnight at roomtemperature. 250 ml of water is added, the organic phase is separated,and it is washed twice in each case with 200 ml of water. The organicphase is dried on magnesium sulfate and evaporated to the dry state in avacuum. The residue is chromatographed on silica gel (mobile solvent:chloroform/methanol/aqueous 25% ammonia 10/5/1). The thus obtained1-[1-(4-benzyloxycarbonylphenyl)-2-oxo-piperidin-3-yl]-1,4,7,10-tetraazacyclododecane(27.6 g; 57.5 mmol; 84% of theory) and 60 ml (0.35 mol) ofN-ethyldiisopropylamine in 200 ml of dichloromethane are added to 62.45g (0.2 mol) of 2-(trifluoromethanesulfonyloxy)propanoic acid benzylester (Kitazaki et al., Chem. Pharm. Bull. (1999), 47(3), 360) in 400 mlof dichloromethane, and it is stirred for 6 hours under reflux and thenovernight at room temperature. It is extracted three times with 500 mlof water, the organic phase is dried on magnesium sulfate and evaporatedto the dry state. The residue is chromatographed on silica gel (mobilesolvent: dichloromethane/methanol: 20/1). The fractions that contain theproduct are combined and concentrated by evaporation.

Yield: 39.4 g (71% of theory) of a colorless, crystalline powder

Elementary analysis:

Cld.: C, 70.86; H, 6.99; N, 7.25.

Fnd.: C 71.11; H, 6.81; N, 7.17.

c)10-[1-(4-Carboxyphenyl)-2-oxo-piperidin-3-yl]-1,4,7-α,α′,α″-trimethyl-1,4,7-tris(carboxymethyl)-1,4,7,10-tetraazacyclododecane

29.0 g (30 mmol) of the title compound of Example 27b is dissolved in400 ml of isopropanol, mixed with 40 ml of water, and 3 g of palladiumcatalyst (10% Pd/C) is added. It is hydrogenated for 8 hours at 50° C.Catalyst is filtered out, and the filtrate is evaporated to the drystate in a vacuum.

Yield: 18.1 g (quantitative) of a colorless powder

Elementary analysis:

Cld.: C, 57.51; H, 7.16; N, 11.56.

Fnd.: C, 57.72; H, 7.11; N, 11.50.

d) Gd Complex of10-[1-(4-carboxyphenyl)-2-oxo-piperidin-3-yl]-1,4,7-α,α′,α″-trimethyl-1,4,7-tris(carboxymethyl)-1,4,7,10-tetraazacyclododecane

12.1 g (20 mmol) of the ligand that is described in Example 27c isdissolved in 200 ml of water and 80 ml of isopropanol, and it isacidified by adding 5 ml of acetic acid. 3.6 g (10 mmol) of gadoliniumoxide is added, and it is refluxed for 3 hours. After complexing iscompleted, it is set at pH 7.4 again with ammonia and chromatographed onsilica gel (mobile solvent: dichloromethane/methanol/ammonia: 20/20/1).The fractions that contain the product are combined and added via anIR-120® cation exchange column (H⁺ form). The acidic eluate isfreeze-dried.

Yield: 11.4 g (72% of theory) of a colorless powder.

Water content (Karl-Fischer): 7.1%

Elementary analysis (relative to anhydrous substance):

Cld.: C, 45.84; H, 5.31; Gd, 20.69; N, 9.22.

Fnd.: C, 45.99; H, 5.26; Gd, 20.55; N, 9.21.

Example 28 a)10-[1-(4-Benzyloxycarbonylphenyl)-2-oxo-piperidin-3-yl]-1,4,7-α,α′,α″-tris(isopropyl)-1,4,7-tris(benzyloxycarbonylmethyl)-1,4,7,10-tetraazacyclododecane

24.0 g (50 mmol) of1-[1-(4-benzyloxycarbonylphenyl)-2-oxo-piperidin-3-yl]-1,4,7,10-tetraazacyclododecanethat is described in Example 27b as an intermediate product and 60 ml(0.35 mol) of N-ethyldiisopropylamine in 200 ml of dichloromethane areadded to 68.1 g (0.2 mol) of 2-(trifluoromethanesulfonyloxy)-isovalericacid benzyl ester (Walker et al., Tetrahedron (1997), 53(43), 14591) in400 ml of dichloromethane, and it is stirred for 6 hours under refluxand then overnight at room temperature. It is extracted three times with500 ml of water each, the organic phase is dried on magnesium sulfateand evaporated to the dry state. The residue is chromatographed onsilica gel (mobile solvent: dichloromethane/methanol: 20/1). Thefractions that contain the product are combined and concentrated byevaporation.

Yield: 37.8 g (72% of theory) of a colorless, crystalline powder

Elementary analysis:

Cld.: C, 72.04; H, 7.58; N, 6.67.

Fnd.: C, 72.32; H, 7.46; N, 6.59.

b)10-[1-(4-Carboxyphenyl)-2-oxo-piperidin-3-yl]-1,4,7-α,α′,α″-tris(isopropyl)-1,4,7-tris(carboxymethyl)-1,4,7,10-tetraazacyclododecane

31.5 g (30 mmol) of the title compound of Example 28a is dissolved in400 ml of isopropanol, mixed with 40 ml of water, and 3 g of palladiumcatalyst (10% Pd/C) is added. It is hydrogenated for 8 hours at 50° C.Catalyst is filtered out, and the filtrate is evaporated to the drystate in a vacuum.

Yield: 20.7 g (quantitative) of a colorless powder

Elementary analysis:

Cld.: C, 60.94; H, 8.04; N, 10.15.

Fnd.: C, 60.87; H, 8.05; N, 10.11.

c) Gd Complex of10-[1-(4-carboxyphenyl)-2-oxo-piperidin-3-yl]-1,4,7-α,α′,α″-tris(isopropyl)-1,4,7-tris(carboxymethyl)-1,4,7,10-tetraazacyclododecane

13.8 g (20 mmol) of the ligand that is described in Example 28b isdissolved in 200 ml of water and 80 ml of isopropanol, and it isacidified by adding 5 ml of acetic acid. 3.6 g (10 mmol) of gadoliniumoxide is added, and it is refluxed for 3 hours. After complexing iscompleted, it is set at pH 7.4 with ammonia and chromatographed onsilica gel (mobile solvent: dichloromethane/methanol/ammonia: 20/20/1).The fractions that contain the product are combined and added via anIR-120® cation exchange column (H⁺ form). The acidic eluate isfreeze-dried.

Yield: 12.0 g (68% of theory) of a colorless powder.

Water content (Karl-Fischer): 7.5%

Elementary analysis (relative to anhydrous substance):

Cld.: C, 49.80; H, 6.21; Gd, 18.63; N, 8.30.

Fnd.: C, 49.99; H, 6.17; Gd, 18.51; N, 8.21.

The Dy complex of10-[1-(4-carboxyphenyl)-2-oxo-piperidin-3-yl]-1,4,7-α,α′,α″-tris(isopropyl)-1,4,7-tris(carboxymethyl)-1,4,7,10-tetraazacyclododecaneis analogously obtained with use of 13.8 g (20 mmol) of the ligand thatis described in Example 28b and 3.73 g (10 mmol) of dysprosium oxideinstead of gadolinium oxide.

Yield: 12.4 g (70% of theory) of a colorless powder.

Water content (Karl-Fischer): 7.5%

Elementary analysis (relative to anhydrous substance):

Cld.: C, 49.50; H, 6.17; Dy, 19.13; N, 8.25.

Fnd.: C, 49.77; H, 6.18; Dy, 18.89; N, 8.27.

Example 29 a)10-[1-(4-Benzyloxycarbonylphenyl)-2-oxo-piperidin-3-yl]-1,4,7-α,α′,α″-tris(cyclohexyl)-1,4,7-tris(benzyloxycarbonylmethyl)-1,4,7,10-tetraazacyclododecane

24.0 g (50 mmol) of1-[1-(4-benzyloxycarbonylphenyl)-2-oxo-piperidin-3-yl]-1,4,7,10-tetraazacyclododecanethat is described in Example 27b as an intermediate product and 60 ml(0.35 mol) of N-ethyldiisopropylamine in 200 ml of dichloromethane areadded to 76.1 g (0.2 mol) of2-(trifluoromethanesulfonyloxy)-2-cyclohexylacetic acid benzyl ester(Qabar et al., Tetrahedron Letters (1998), 39(33), 5895) in 400 ml ofdichloromethane, and it is stirred for 6 hours under reflux and thenovernight at room temperature. It is extracted three times with 50 ml ofwater each, the organic phase is dried on magnesium sulfate andevaporated to the dry state. The residue is chromatographed on silicagel (mobile solvent: dichloromethane/methanol: 20/1). The fractions thatcontain the product are combined and concentrated by evaporation.

Yield: 40.9 g (70% of theory) of a colorless, crystalline powder

Elementary analysis:

Cld.: C, 73.88; H, 7.84; N, 5.98.

Fnd.: C, 74.12; H, 7.69; N, 5.89.

b)10-[1-(4-Carboxyphenyl)-2-oxo-piperidin-3-yl]-1,4,7-α,α′,α″-tris(cyclohexyl)-1,4,7-tris(carboxymethyl)-1,4,7,10-tetraazacyclododecane

35.1 g (30 mmol) of the title compound of Example 29a is dissolved in400 ml of isopropanol, mixed with 40 ml of water, and 3 g of palladiumcatalyst (10% Pd/C) is added. It is hydrogenated for 8 hours at 50° C.Catalyst is filtered out, and the filtrate is evaporated to the drystate.

Yield: 24.3 g (quantitative) of a colorless powder

Elementary analysis:

Cld.: C, 65.24; H, 8.34; N, 8.65.

Fnd.: C, 65.48; H, 8.22; N, 8.60.

c) Gd Complex of10-[1-(4-carboxyphenyl)-2-oxo-piperidin-3-yl]-1,4,7-α,α′,α″-tris(cyclohexyl)-1,4,7-tris(carboxymethyl)-1,4,7,10-tetraazacyclododecane

16.2 g (20 mmol) of the ligand that is described in Example 29b isdissolved in 150 ml of water and 150 ml of isopropanol, and it isacidified by adding 5 ml of acetic acid. 3.6 g (10 mmol) of gadoliniumoxide is added, and it is refluxed for 8 hours. After complexing iscompleted, it is set at pH 7.4 again with ammonia and chromatographed onsilica gel (mobile solvent: dichloromethane/methanol/ammonia: 20/20/1).The fractions that contain the product are combined and evaporated tothe dry state. The residue is taken up with formic acid and evaporatedto the dry state several times with the addition of dichloromethane andthen dried in a vacuum until a constant weight is reached.

Yield: 13.6 g (68% of theory) of a colorless powder.

Water content (Karl-Fischer): 7.5%

Elementary analysis (relative to anhydrous substance):

Cld.: C, 54.81; H, 6.69; Gd, 16.31; N, 7.26.

Fnd.: C, 55.11; H, 6.57; Gd, 16.09; N, 7.24.

Example 30 a)1,7-Bis(benzyloxycarbonyl)-4,10-α,α′-dimethyl-4,10-bis(t-butoxycarbonylmethyl)-1,4,7,10-tetraazacyclododecane

13.8 g (100 mmol) potassium carbonate and 15.3 g (55 mmol)2-(trifluoromethane-sulfonyloxy)propane acid t-butyl ester (Decicco etal., Journal of Organic Chemistry 1995, 60, 4782) are added to 11.01 g(25 mmol) 1,7-bis(benzyloxycarbonyl)-1,4,7,10-tetraazacyclododecane(Kovacs et al., Synthesis 1997, 759), dissolved in 150 ml oftetrahydrofurane and 15 ml of water, and it is stirred over night atroom temperature. After evaporation to the dry state, the residue isdistributed between 100 ml dichloromethane and 100 ml of water, theorganic phase is separated and it is washed twice in each case with 50ml of water. The organic phase is dried on magnesium sulfate andevaporated to the dry state in a vacuum. The residue is chromatographedon silica gel (mobile solvent: chloroform/methanol/aqu. 25%ammonia=10/5/1). The fractions that contain the product are combined andconcentrated by evaporation.

Yield: 17.4 g (86% of theory) of a colorless powder

Elementary analysis:

Cld.: C, 65.49; H, 8.10; N, 8.04.

Fnd.: C, 65.22; H, 8.17; N, 8.20.

b)1,7-α,α′-Dimethyl-1,7-bis(t-butoxycarbonylmethyl)-1,4,7,10-tetraazacyclododecane

13.9 g (20 mmol) of the title compound of example 30a is dissolved in250 ml of isopropanol, mixed with 25 ml of water, and 2 g of palladiumcatalyst (10% Pd/C) is added. It is hydrogenated for 8 hours at 50° C.Catalyst is filtered out, and the filtrate is evaporated to the drystate in a vacuum.

Yield: 8.5 g (quantitative) of a colorless powder

Elementary analysis:

Cld.: C, 61.65; H, 10.35; N, 13.07.

Fnd.: C, 61.17; H, 10.55; N, 12.85.

c)1,7-α,α′-Dimethyl-1,7-bis-(t-butoxycarbonylmethyl)-4-α-isopropyl-4-benzyloxycarbonylmethyl)-1,4,7,10-tetraazacyclododecane

8.51 g (25 mmol) 2-(trifluoromethane-sulfonyloxy)-isovaleric acid benzylester (Walker et al., Tetrahedron (1997), 53(43), 14591) in 60 ml ofdichloromethane is added to 8.5 g (20 mmol) of that in example 30bdescribed1,7-α,α′-dimethyl-1,7-bis-(t-butoxycarbonylmethyl)-1,4,7,10-tetraazacyclododecaneand 6.86 ml (40 mmol) N-ethyldiisopropylamine in 200 ml ofdichloromethane and it is stirred for 6 hours under reflux and then overnight at room temperature. It is extracted three times in each case with50 ml of water, the organic phase is dried on magnesium sulfate andevaporated to the dry state. The residue is chromatographed on silicagel (mobile solvent: dichloromethane/methanol: 20/1). The fractions thatcontain the product are combined and concentrated by evaporation.

Yield: 8.8 g (71% of theory) of a colorless powder

Elementary analysis:

Cld.: C, 65.99; H, 9.45; N, 9.05.

Fnd.: C, 66.08; H, 9.25; N, 8.85.

d)10-[4-(Benzyloxycarbonyl)-1-methyl-2-oxo-3-azabutyl]-1,7-α,α′-dimethyl-1,7-bis(t-butoxycarbonylmethyl)-4-α-isopropyl-4-benzyloxycarbonylmethyl)-1,4,7,10-tetraazacyclododecane

2.5 g (8.11 mmol) 2-bromopropionylglycin-benzyl ester (example 1e of WO98/24774) is added to 4.33 g (7 mmol) of that in example 30c described1,7-α,α′-dimethyl-1,7-bis(t-butoxycarbonylmethyl)-4-α-isopropyl-4-benzyloxycarbonylmethyl)-1,4,7,10-tetraazacyclododecane,dissolved in 50 ml of chloroform, and it is stirred at 50° C. overnight. 50 ml of water are added, the organic phase is separated and itis washed twice in each case with 20 ml of water. The organic phase isdried on magnesium sulfate and evaporated to the dry state in a vacuum.The residue is chromatographed on silica gel (mobile solvent:dichloromethane/methanol: 20/1). The fractions that contain the productare combined and concentrated by evaporation.

Yield: 3.1 g (53% of theory) of a colorless powder

Elementary analysis:

Cld.: C, 65.92; H, 8.54; N, 8.36.

Fnd.: C, 65.77; H, 8.62; N, 8.20.

e) Gd-complex of10-(4-carboxy-1-methyl-2-oxo-3-azabutyl)-1,7-α,α′-dimethyl-1,7-bis-(carboxymethyl)-4-α-isopropyl-4-carboxymethyl)-1,4,7,10-tetraazacyclododecane

2.5 g (3 mmol) of the title compound of example 30d is dissolved in 100ml of isopropanol, mixed with 5 ml of water and after addition of aspatula tip of palladium catalyst (10% Pd/C), it is hydrogenated for 8hours at room temperature. Catalyst is filtered out, and the filtrate isevaporated to the dry state in a vacuum. The residue is dissolved in 25ml of trifluoroacetic acid without further purification and it isstirred for 2 hours at room temperature. The deprotected product isprecipitated by the addition of diethylether, exhausted and dried in avacuum. The residue is dissolved in 20 ml of water and 2 ml ofisopropanol and the pH is adjusted to 5. 543 mg (1.5 mmol) gadoliniumoxide is added and it is refluxed for 3 hours. After the complexation isfinished, the pH is adjusted to 7.4 with ammonia and it ischromatographed on silica gel (mobile solvent: dichloromethanemethanol/ammonia: 20/20/1). The fractions that contain the product arecombined and added via an IR-120®-cation exchange column (H⁺-form). Theacidic eluate is freeze-dried.

Yield: 1.44 g (62% of theory) of a colorless powder. Water content(Karl-Fischer): 9.3%

Elementary analysis (referenced to the anhydrous substance):

Cld.: C, 41.19; H, 5.76; Gd, 22.47; N, 10.01.

Fnd.: C, 40.88; H, 5.88; Gd, 22.11; N, 9.79.

The dysprosium complex is obtained analog to this by using 559 mg (1.5mmol) dysprosium oxide instead of gadolinium oxide:

Yield: 1.44 g (62% of theory) of a colorless powder. Water content(Karl-Fischer): 9.0%

Elementary analysis (referenced to the anhydrous substance):

Cld.: C, 40.88; H, 5.72; Dy 23.05; N, 9.93

Fnd.: C, 40.58; H, 5.90; Dy, 22.73; N, 9.86.

Example 31 a)1,7-Bis(benzyloxycarbonyl)-4,10-α,α′-diisopropyl-4,10-bis(t-butoxycarbonylmethyl)-1,4,7,10-tetraazacyclododecane

13.8 g (100 mmol) potassium carbonate and 16.85 g (55 mmol)2-(trifluoromethane-sulfonyloxy)-isovaleric acid t-butyl ester(Semmelhack et al., Tetrahedron Letters 34, 1395 (1993)) are added to11.01 g (25 mmol)1,7-bis(benzyloxycarbonyl)-1,4,7,10-tetraazacyclododecane (Kovacs etal., Synthesis 1997, 759), dissolved in 150 ml of tetrahydrofurane and15 ml of water and it is stirred over night at room temperature. It isevaporated to the dry state in a vacuum and the residue is distributedbetween 100 ml of dichloromethane and 100 ml of water, the organic phaseis separated and washed twice with each 50 ml of water. The organicphase is dried on magnesium sulfate and evaporated to the dry state in avacuum. The residue is chromatographed on silica gel (mobile solvent:chloroform/methanol/aqu. 25% ammonia=10/5/1). The fractions that containthe product are combined and concentrated by evaporation.

Yield: 13.9 g (74% of theory) of a colorless powder.

Elementary analysis:

Cld.: C, 66.99; H, 8.57; N, 7.44.

Fnd.: C, 66.58; H, 8.72; N, 7.22.

b)1,7-α,α′-Diisopropyl-1,7-bis(t-butoxycarbonylmethyl)-1,4,7,10-tetraazacyclododecane

15.06 g (20 mmol) of the title compound of example 31a is dissolved in250 ml of isopropanol, 25 ml of water is added and 2 g palladiumcatalyst (10% Pd/C) are added. It is hydrogenated for 8 hours at 50° C.Catalyst is filtered out, and the filtrate is evaporated to the drystate in a vacuum.

Yield: 9.7 g (quantitative) of a colorless powder.

Elementary analysis:

Cld.: C, 64.43; H, 10.81; N, 11.56.

Fnd.: C, 64.30; H, 10.94; N, 11.31.

c)1,7-α,α′-Diisopropyl-1,7-bis(t-butoxycarbonylmethyl)-4-α-methyl-4-benzyloxycarbonyl-methyl-1,4,7,10-tetraazacyclododecane

8.18 g (25 mmol) 2-(trifluoromethane-sulfonyloxy)propanoic acid-benzylester (Kitazaki et al., Chem. Pharm. Bull. (1999), 47(3), 360) in 60 mlof dichloromethane is added to 8.5 g (20 mmol) of that in example 31bdescribed1,7-α,α′-diisopropyl-1,7-bis(t-butoxycarbonylmethyl)-1,4,7,10-tetraazacyclododecaneand 6.86 ml (40 mmol) N-ethyldiisopropylamine in 200 ml ofdichloromethane, and it is stirred for 6 hours under reflux and thenover night at room temperature. It is extracted three times in each casewith 50 ml of water, the organic phase is dried on magnesium sulfate andevaporated to the dry state. The residue is chromatographed on silicagel (mobile solvent: dichloromethane/methanol: 20/1). The fractions thatcontain the product are combined and concentrated by evaporation.

Yield: 9.8 g (76% of theory) of a colorless powder.

Elementary analysis:

Cld.: C, 66.84; H, 9.66; N, 8.66.

Fnd.: C, 66.58; H, 9.82; N, 8.63.

d)10-[4-(Benzyloxycarbonyl)-1-methyl-2-oxo-3-azabutyl]-1,7-α,α′-diisopropyl-1,7-bis(t-butoxycarbonylmethyl)-4-α-methyl-4-benzyloxycarbonylmethyl-1,4,7,10-tetraazacyclododecane

2.5 g (8.11 mmol) 2-bromopropionylglycin-benzyl ester (example 1e of WO98/24774) is added to 4.53 g (7 mmol) of that in example 31c described1,7-α,α′-diisopropyl-1,7-bis(t-butoxycarbonylmethyl)-4-α-methyl-4-benzyloxycarbonylmethyl)-1,4,7,10-tetraazacyclododecane,dissolved in 50 ml of chloroform, and it is stirred at 50° C. overnight. 50 ml of water is added, the organic phase is separated andwashed twice in each case with 20 ml of water. The organic phase isdried on magnesium sulfate and evaporated to the dry state in a vacuum.The residue is chromatographed on silica gel (mobile solvent:dichloromethane/methanol: 20/1). The fractions that contain the productare combined and concentrated by evaporation.

Yield: 3.6 g (59% of theory) of a colorless powder.

Elementary analysis:

Cld.: C, 66.56; H, 8.73; N, 8.09.

Fnd.: C, 66.26; H, 8.98; N, 7.91.

e) Gd-complex of10-(4-carboxy-1-methyl-2-oxo-3-azabutyl)-1,7-α,α′-diisopropyl-1,7-bis(carboxymethyl)-4-α-methyl-4-carboxymethyl-1,4,7,10-tetraazacyclododecane

2.6 g (3 mmol) of the title compound of example 31d is dissolved in 100ml of isopropanol, 5 ml of water is added and after addition of aspatula tip of palladium catalyst (10% Pd/C), it is hydrogenated for 8hours at room temperature. Catalyst is filtered out, and the filtrate isevaporated to the dry state in a vacuum. The residue is dissolved in 25ml of trifluoroacetic acid without further purification and it isstirred for 2 hours at room temperature. The deprotected product isprecipitated by addition of diethylether, exhausted and dried in avacuum. The residue is dissolved in 20 ml of water and 2 ml ofisopropanol and the pH is adjusted to 5.543 mg (1.5 mmol) gadoliniumoxide is added and it is refluxed for 3 hours. After the complexation isfinished, the pH is adjusted to 7.4 with ammonia and it ischromatographed on silica gel (mobile solvent:dichloromethane/methanol/ammonia: 20/20/1). The fractions that containthe product are combined and added via an IR-120®-cation exchange column(H⁺-form). The acidic eluate is freeze-dried.

Yield: 1.55 g (64% of theory) of a colorless powder.

Water content (Karl-Fischer): 10.0%

Elementary analysis (referenced to the anhydrous substance):

Cld.: C, 42.90; H, 6.09; Gd, 21.60; N, 9.62.

Fnd.: C, 42.75; H, 5.93; Gd, 21.21; N, 9.54.

Example 32 a)1,7-α,α′-dimethyl-1,7-bis(t-butoxycarbonylmethyl)-4-α-cyclohexyl-4-benzyloxycarbonylmethyl-1,4,7,10-tetraazacyclododecane

9.51 g (25 mmol) 2-(trifluoromethane-sulfonyloxy)-2-cyclohexyl aceticacid-benzyl ester (Qabar et al., Tetrahedron Letters (1998), 39(33),5895) in 60 ml of dichloromethane is added to 8.5 g (20 mmol) of that inexample 30b described1,7-α,α′-dimethyl-1,7-bis(t-butoxycarbonylmethyl)-1,4,7,10-tetraazacyclododecaneand 6.86 ml (40 mmol) of N-ethyldiisopropylamine in 200 mldichloromethane and it is stirred for 6 hours under reflux and then overnight at room temperature. It is extracted three times with in each case50 ml of water, the organic phase is dried on magnesium sulfate andevaporated to the dry state. The residue is chromatographed on silicagel (mobile solvent: dichloromethane/methanol: 20/1). The fractions thatcontain the product are combined and concentrated by evaporation.

Yield: 9.1 g (69% of theory) of a colorless powder.

Elementary analysis (referenced to the anhydrous substance):

Cld.: C, 67.44; H, 9.48; N, 8.50.

Fnd.: C, 67.28; H, 9.66; N, 8.31.

b)10-[4-(Benzyloxycarbonyl)-1-methyl-2-oxo-3-azabutyl]-1,7-α,α′-dimethyl-1,7-bis(t-butoxycarbonylmethyl)-4-α-cyclohexyl-4-benzyloxycarbonylmethyl-1,4,7,10-tetraazacyclododecane

2.5 g (8.11 mmol) 2-bromopropionylglycin-benzyl ester (example 1e of WO98/24774) is added to 4.61 g (7 mmol) of that in example 32a described1,7-α,α′-dimethyl-1,7-bis(t-butoxycarbonylmethyl)-4-α-cyclohexyl-4-benzyloxycarbonylmethyl-1,4,7,10-tetraazacyclododecane,dissolved in 50 ml of chloroform, and it is stirred over night at 50° C.50 ml of water is added, the organic phase is separated and it is washedtwice in each case with 20 ml of water. The organic phase is dried onmagnesium sulfate and evaporated to the dry state in a vacuum. Theresidue is chromatographed on silica gel (mobile solvent:dichloromethane/methanol: 20/1). The fractions that contain the productare combined and concentrated by evaporation.

Yield: 3.5 g (57% of theory) of a colorless powder

Elementary analysis:

Cld.: C, 67.02; H, 8.61; N, 7.97.

Fnd.: C, 66.86; H, 8.62; N, 8.11.

c) Gd complex of the10-(4-carboxy-1-methyl-2-oxo-3-azabutyl)-1,7-α,α′-dimethyl-1,7-bis(carboxymethyl)-4-α-cyclohexyl-4-carboxymethyl-1,4,7,10-tetraazacyclododecane

2.6 g (3 mmol) of the title compound of example 32b is dissolved in 100ml of isopropanol, 5 ml of water is added and after addition of aspatula tip of palladium catalyst (10% Pd/C), it is hydrogenated for 8hours at room temperature. Catalyst is filtered off and the filtrate isevaporated to the dry state in a vacuum. The residue is dissolved in 25ml of trifluoroacetic acid without further purification and it isstirred for two hours at room temperature. The deprotected product isprecipitated by addition of diethylether, exhausted and dried in avacuum. The residue is dissolved in 20 ml of water and 2 ml ofisopropanol and the pH is adjusted to 5. 543 mg (1.5 mmol) gadoliniumoxide are added and it is refluxed for 3 hours. After the complexationis finished, the pH is adjusted to 7.4 with ammonia and it ischromatographed on silica gel (mobile solvent:dichloromethane/methanol/ammonia: 20/20/1). The fractions that containthe product are combined and added via an IR-120®-cation exchange column(H⁺-form). The acidic eluate is freeze-dried.

Yield: 1.56 g (64% of theory) of a colorless powder

Water content (Karl-Fischer): 9.1%

Elementary analysis (referenced to the anhydrous substance):

Cld.: C, 43.83; H, 5.99; Gd, 21.25; N, 9.46.

Fnd.: C, 43.62; H, 6.18; Gd, 21.04; N, 9.31.

Example 33 a) 10-[α-(4-(Ethoxycarbonylmethoxy)phenyl)-methoxycarbonylmethyl]-1,7-α,α′-dimethyl-1,7-bis(t-butoxycarbonylmethyl)-4-α-isopropyl-4-benzyloxycarbonylmethyl-1,4,7,10-tetraazacyclododecane

2.69 g (8.11 mmol) of that in example 5b describedα-bromo-4-(ethoxycarbonylmethoxy)-phenylacetic acid methyl ester isadded to 4.33 g (7 mmol) of that in example 30c described1,7-α,α′-dimethyl-1,7-bis-(t-butoxycarbonylmethyl)-4-α-isopropyl-4-benzyloxycarbonylmethyl-1,4,7,10-tetraazacyclododecane,dissolved in 50 ml of chloroform, and it is stirred over night at 50° C.50 ml of water is added, the organic phase is separated and it is washedtwice in each case with 20 ml of water. The organic phase is dried onmagnesium sulfate and evaporated to the dry state in a vacuum. Theresidue is chromatographed on silica gel (mobile solvent:dichloromethane/methanol: 20/1). The fractions that contain the productare combined and concentrated by evaporation.

Yield: 3.5 g (58% of theory) of a colorless powder

Elementary analysis:

Cld.: C, 64.95; H, 8.35; N, 6.45.

Fnd.: C, 64.73; H, 8.22; N, 6.58.

b) Gd-complex of the10-[α-(4-carboxymethoxyphenyl)-carboxymethyl]-1,7-α,α′-dimethyl-1,7-bis(carboxymethyl)-4-α-isopropyl-4-carboxymethyl-1,4,7,10-tetraazacyclododecane

2.6 g (3 mmol) of the title compound of example 33a is dissolved in 100ml of isopropanol, 5 ml of water is added and after addition of aspatula tip of palladium catalyst (10% Pd/C), it is hydrogenated for 8hours at 50° C. Catalyst is filtered off, the filtrate is evaporated tothe dry state in a vacuum. The residue is treated with 40 ml of a 2Nsodium hydroxide solution and 40 ml tetrahydrofuran, without furtherpurification and it is stirred for 5 days at 40° C. Afterwards the pH isadjusted to 7 with Amberlite IR-120® (H⁺-form) and acidified by additionof 1 ml acetic acid. Then 543 mg (1.5 mmol) gadolinium oxide is addedand it is refluxed for 3 hours. After the complexation is finished thepH is adjusted to 7.4 with ammonia and it is chromatographed on silicagel (mobile solvent: dichloromethane/methanol/ammonia: 20/20/1). Thefractions that contain the product are combined and added via anIR-120®-cation exchange column (H⁺-form). The acidic eluate isfreeze-dried.

Yield: 1.82 g (71% of theory) of a colorless powder

Water content (Karl-Fischer): 8.9%

Elementary analysis (referenced to the anhydrous substance):

Cld.: C, 44.72; H, 5.31; Gd, 20.19; N, 7.19.

Fnd.: C, 44.88; H, 5.21; Gd, 20.01; N, 6.95.

Example 34 a)10-[4-(Benzyloxycarbonyl)-2-oxo-3-azabutyl]-1,7-α-α′-diisopropyl-1,7-bis(t-butoxycarbonylmethyl)-4-α-methyl-4-benzyloxycarbonylmethyl-1,4,7,10-tetraazacyclododecane

2.32 g (8.11 mmol) 2-bromoacetylglycin-benzyl ester (Teger-Nilsson etal., WO 93/11152, page 38) is added to 4.53 g (7 mmol) of that inexample 31c described1,7-α-α′-diisopropyl-1,7-bis(t-butoxycarbonylmethyl)-4-α-methyl-4-benzyloxycarbonylmethyl-1,4,7,10-tetraazacyclododecane,dissolved in 50 ml of chloroform, and it is stirred over night at 50° C.50 ml of water is added, the organic phase is separated and washed twicein each case with 20 ml of water. The organic phase is dried onmagnesium sulfate and evaporated to the dry state in a vacuum. Theresidue is chromatographed on a silica gel (mobile solvent:dichloromethane/methanol: 20/1). The fractions that contain the productare combined and concentrated by evaporation.

Yield: 4.8 g (81% of theory) of a colorless powder.

Elementary analysis:

Cld.: C, 66.25; H, 8.64; N, 8.22.

Fnd.: C, 66.00; H, 8.56; N, 8.31.

b) Gd-complex of the10-(4-carboxy-2-oxo-3-azabutyl)-1,7-α,α′-diisopropyl-1,7-bis(carboxymethyl)-4-α-methyl-4-carboxymethyl-1,4,7,10-tetraazacyclododecane

2.55 g (3 mmol) of the title compound of example 34a is dissolved in 100ml of isopropanol, 5 ml of water is added and after addition of aspatula tip palladium catalyst (10% Pd/C) it is hydrogenated for 8 hoursa room temperature. Catalyst is filtered off, and the filtrate isevaporated to the dry state in a vacuum. The residue is dissolved in 25ml of trifluoroacetic acid without further purification and it isstirred for 2 hours at room temperature. The deprotected product isprecipitated by addition of diethylether, exhausted and dried in avacuum. The residue is dissolved in 20 ml of water and 2 ml ofisopropanol and the pH is adjusted to 5. 543 mg (1.5 mmol) gadoliniumoxide is added and it is refluxed for 3 hours. After the complexation isfinished the pH is adjusted to 7.4 with ammonia and it ischromatographed on silica gel (mobile solvent:dichloromethane/methanol/ammonia: 20/20/1). Fractions that contain theproduct are combined and added via an IR-120®-cation exchange column(H⁺-form). The acidic eluate is freeze-dried.

Yield: 1.51 g (64% of theory) of a colorless powder.

Water content (Karl-Fischer): 9.0%

Elementary analysis (referenced to the anhydrous substance):

Cld.: C, 42.06; H, 5.93; Gd, 22.03; N, 9.81.

Fnd.: C, 41.95; H, 5.99; Gd, 21.77; N, 9.62.

Example 35 a)10-[4-(Benzyloxycarbonyl)-2-oxo-3-azabutyl]-1,7-α-α′-dimethyl-1,7-bis(t-butoxycarbonylmethyl)-4-α-cyclohexyl-4-benzyloxycarbonylmethyl-1,4,7,10-tetraazacyclododecane

2.32 g (8.11 mmol) 2-bromoacetylglycin-benzyl ester (Teger-Nilsson etal., WO 93/11152, page 38) is added to 4.61 g (7 mmol) of that inexample 32a described1,7-α,α′-dimethyl-1,7-bis(t-butoxycarbonylmethyl)-4-α-cyclohexyl-4-benzyloxycarbonylmethyl-1,4,7,10-tetraazacyclododecane,dissolved in 50 ml of chloroform, and it is stirred over night at 50° C.50 ml of water is added, the organic phase is separated and washed twicein each case with 20 ml of water. The organic phase is dried onmagnesium sulfate and evaporated to the dry state in a vacuum. Theresidue is chromatographed on silica gel (mobile solvent:dichloromethane/methanol 20/1). The fractions that contain the productare combined and concentrated by evaporation.

Yield: 4.35 g (72% of theory) of a colorless powder.

Elementary analysis:

Cld.: C, 66.72; H, 8.51; N, 8.10.

Fnd.: C, 66.43; H, 8.77; N, 8.02.

b) Gd-complex of the10-(4-carboxy-2-oxo-3-azabutyl)-1,7-α,α′-dimethyl-1,7-bis(carboxymethyl)-4-α-cyclohexyl-4-carboxymethyl-1,4,7,10-tetraazacyclododecane

2.6 g (3 mmol) of the title compound of example 35a is dissolved in 100ml of isopropanol, 5 ml of water is added and after the addition of aspatula tip of palladium catalyst (10% Pd/C) it is hydrogenated for 8hours at room temperature. Catalyst is filtered out, the filtrate isevaporated to the dry state in a vacuum. The residue is dissolved in 25ml of trifluoroacetic acid without further purification and it isstirred for 2 hours at room temperature. The deprotected product isprecipitated by the addition of diethylether, exhausted and dried in avacuum. The residue is dissolved in 20 ml of water and 2 ml ofisopropanol and the pH is adjusted to 5.543 mg (1.5 mmol) gadoliniumoxide is added and it is refluxed for 3 hours. After the complexation isfinished, the pH is adjusted to 7.4 with ammonia and it ischromatographed on silica gel (mobile solvent:dichloromethane/methanol/ammonia: 20/20/1). The fractions that containthe product are combined and added via an IR-120®-cation exchange column(H⁺-form). The acidic eluate is freeze-dried.

Yield: 1.74 g (73% of theory) of a colorless powder.

Water content (Karl-Fischer): 8.8%

Elementary analysis (referenced to the anhydrous substance):

Cld.: C, 43.02; H, 5.83; Gd, 21.66; N, 9.65.

Fnd.: C, 42.87; H, 6.05; Gd, 21.29; N, 9.55.

Examples 36-108

Examples 36-108 describe conjugates of the above-described gadoliniumcomplexes with biomolecules. The conjugates were produced according tothe following general operating instructions I-IV. The results aresummarized in Table 1. Here, “AAV” stands for general operatinginstructions, “ACTH” stands for adrenocorticotropic hormone, and “RP-18”refers to a “reversed phase” stationary chromatography phase. The numberof complexes per biomolecule was determined by means of ICP (inductivelycoupled plasma atomic emission spectroscopy).

General Operating Instructions (AAV) I: Albumin-Amide Conjugates

3 mmol of the Gd complex acid is dissolved in 15 ml of DMF, mixed with380 mg (3.3 mmol) of N-hydroxysuccinimide and 681 mg ofdicyclohexylcarbodiimide while being cooled with ice, and preactivatedfor 1 hour in ice. The active ester mixture is added in drops within 30minutes in a solution of 16.75 g (0.25 mmol) of bovine serum albumin(BSA) or human serum albumin (HSA) in 150 ml of phosphate buffer (pH7.4) and stirred for 2 hours at room temperature. The batch solution isfiltered, the filtrate is ultrafiltered with an AMICON® YM30 (cut-off30,000 Da), the retentate is chromatographed on a Sephadex® G50-column,and the product fractions are freeze-dried.

General Operating Instructions (AAV) II: Albumin-Maleimide Conjugates

0.0438 mmol of the Gd-complex maleimide in 1 ml of DMF is added to 0.84g (0.0125 mmol) of bovine serum albumin (BSA), dissolved in 15 ml ofphosphate buffer (pH 7.4), and it is stirred for one hour at roomtemperature. The batch solution is filtered, the filtrate isultrafiltered with an AMICON® YM30 (cut-off 30,000 Da), the retentate ischromatographed on a Sephadex® G50 column, and the product fractions arefreeze-dried.

General Operating Instructions (AAV) III: Production of Amide Conjugates

3 mmol of the Gd-complex acid is dissolved in 15 ml of DMF, mixed with380 mg (3.3 mmol) of N-hydroxysuccinimide and 681 mg ofdicyclohexylcarbodiimide while being cooled with ice, and preactivatedfor 1 hour in ice. The active ester mixture is added in drops to asolution of 2.5 mmol of amine components in 15-150 ml of DMF and stirredovernight at room temperature. The batch solution is filtered andchromatographed on silica gel.

General Operating Instructions (AAV) IV: Production of Maleimido-SHConjugates

3 mmol of the Gd-complex maleimide in 15 ml of DMF is added in drops to2.5 mmol of SH components in 15-150 ml of DMF, and it is stirred for onehour at room temperature. The batch solution is chromatographed onsilica gel. TABLE 1 Edukt Gd- Anzahl Komplexe Komplex pro BiomolekülAusbeute Beispiel (Beispiel Nr.) konjugiert mit (Herkunft) AAV (ICP)Bemerkungen (%) 36 1 BSA Sigma I 3.7 — quant. 37 2 BSA Sigma I 6.1 —quant. 38 3 BSA Sigma I 2.9 — quant. 39 4 BSA Sigma I 3.5 — quant. 40 5BSA Sigma I 4.2 — quant. 41 6 BSA Sigma I 6.5 — quant. 42 7 BSA Sigma I5.0 — quant. 43 16 BSA Sigma II 0.71 — quant. 44 17 BSA Sigma II 0.55 —quant. 45 8 BSA Sigma I 3.0 — quant. 46 9 BSA Sigma I 4.7 — quant. 47 10BSA Sigma I 5.1 — quant. 48 11 BSA Sigma I 2.7 — quant. 49 12 BSA SigmaI 4.0 — quant. 50 13 BSA Sigma I 3.3 — quant. 51 14 BSA Sigma I 5.8 —quant. 52 15 BSA Sigma I 4.6 — quant. 53 18 BSA Sigma I 3.7 — quant. 5419 BSA Sigma I 4.1 — quant. 55 20 BSA Sigma I 2.8 — quant. 56 21 BSASigma I 3.5 — quant. 57 22 BSA Sigma I 3.3 — quant. 58 23 BSA Sigma I2.9 — 59 24 BSA Sigma I 4.0 — quant. 60 25 BSA Sigma I 3.5 — quant. 6126 BSA Sigma I 3.0 — quant. 62 27 BSA Sigma I 3.9 — quant. 63 28 BSASigma I 3.1 — quant. 64 29 BSA Sigma I 3.4 — quant. 65 11 (D-Lys16)-ACTH(1-24 human) BACHEM I 2.0 — quant. 66 12 ACTH (1-17) BACHEM I 1.7 —quant. 67 14 H-β-Ala-Phe BACHEM III 1.0 wurde an RP-18 95 gereinigt 68 8Anti-Inflamatory Peptide 2 BACHEM I 1.0 — quant. 69 9 L-Carnosin BACHEMIII 1.0 wurde an RP-18 97 gereinigt 70 16 Homoglutathion BACHEM IV 1.0wurde an RP-18 94 gereinigt 71 17 Guanyl-Cys-OH BACHEM IV 1.0 wurde anRP-18 93 gereinigt 72 8 H-DL-d-Hydroxy-DL-Lys-OH BACHEM III 1.0 wurde anRP-18 85 gereinigt 73 7 H-β-Ala-Lys-OH BACHEM III 1.0 wurde an RP-18 87gereinigt 74 16 H-Arg-Gly-Asp-Cys-OH BACHEM III 1.0 wurde an RP-18 91gereinigt 75 9 H-Asp-Leu-Trp-Gln-Lys-OH BACHEM III 1.0 wurde an RP-18 94gereinigt 76 12 H-Ala-His-Lys-OH BACHEM III 2.0 wurde an RP-18 91gereinigt 77 13 Endothelin-2 (Human) BACHEM I 0.87 — quant. 78 14 HumanSerumalbumin BACHEM I 5.1 — quant. 79 7 Human Serumalbumin BACHEM I 3.1— quant. 80 8 Human Serumalbumin BACHEM I 2.3 — quant. 81 17Thioguanosin Aldrich IV 1.0 wurde an RP-18 96 gereinigt 82 56-Aminopenicilinsäure Aldrich III 1.0 wurde an RP-18 92 gereinigt 83 114-Aminopteroylglutaminsäure Aldrich III 1.0 wurde an RP-18 65 gereinigt84 4 2-Amino-purinthiol Aldrich IV 1.0 wurde an RP-18 94 gereinigt 85 125-Azacytidin Aldrich III 1.0 wurde an RP-18 96 gereinigt 86 174,5-Diamino-2,6- Aldrich IV 1.0 wurde an RP-18 71 dimercaptopyrimidingereinigt 87 13 Mitomycin C Aldrich III 1.0 wurde an RP-18 81 gereinigt88 12 Muraminsäure Aldrich III 1.0 wurde an RP-18 92 gereinigt 89 6Puromycin SIGMA III 1.0 wurde an RP-18 90 gereinigt 90 11 DoxorubicinSIGMA III 1.0 wurde an RP-18 89 gereinigt 91 12 Spectinomycin SIGMA III1.0 wurde an RP-18 88 gereinigt 92 4 Streptomycin SIGMA III 1.0 wurde anRP-18 62 gereinigt 93 14 Neomycin B SIGMA III 1.0 wurde an RP-18 52gereinigt 94 8 Nystatin SIGMA III 1.0 wurde an RP-18 72 gereinigt 95 3Hygromycin SIGMA III 1.0 wurde an RP-18 71 gereinigt 96 2 AmpicillinSIGMA III 1.0 wurde an RP-18 42 gereinigt 97 30 BSA Sigma I 2.1 — quant.98 31 BSA Sigma I 1.8 — quant 99 32 BSA Sigma I 2.7 — quant 100 33 BSASigma I 1.2 — quant 101 34 BSA Sigma I 1.6 — quant 102 35 BSA Sigma I2.5 — quant 103 30 HSA Sigma I 1.9 — quant 104 31 HSA Sigma I 2.5 —quant 105 32 HSA Sigma I 2.2 — quant 106 33 HSA Sigma I 1.5 — Quant 10734 HSA Sigma I 1.7 — quant 108 35 HSA Sigma I 2.4 — quant#[Key to Table 1:]Beispiel = ExampleEdukt Gd-Komplex (Beispiel Nr.) = Gd-Complex Educt (Example No.)konjugiert mit = Conjugated with(Herkunft) = (Origin)Anzahl Komplexe pro Biomolekül = Number of complexes per biomoleculeBemerkungen = RemarksAusbeute (%) = Yield (%)L-Carnosin = L-CarnosineHomoglutathion = Homoglutathionewurde an RP-18 gereinigt = was purified on RP-18Thioguanosin = Thioguanosine6-Aminopenicilinsäure = 6-Aminopenicillic acid4-Aminopteroylglutaminsäure = 4-Aminopteroylglutamic acid2-Amino-purinthiol = 2-amino-purinethiol5-Azacytidin = 5-Azacytidine4,5-Diamino-2,6-dimercaptopyrimidin =4,5-Diamino-2,6-dimercaptopyridimidineMuraminsäure = Muramic acid

Example 109

In this example, the relaxivities of the conjugates from Examples 36-38and 97-108 were compared with the relaxivities of two comparisonsubstances. As comparison substances, Gd-DTPA (1) with the formula:

and Gd-GlyMeDOTA (2) with the formula:

which were reacted in each case with bovine serum albumin (BSA), wereused.

The measurements were made in each case in aqueous solution and inplasma at +37° C. and a frequency of 20 MHz. The results are summarizedin Table 2 below, whereby the indicated relaxivities per mol ofgadolinium were calculated from the measured values: TABLE 2 Gd-KomplexAnzahl Gd/BSA R₁ (H₂O) R₁ (Plasma) Beispiel (aus Beispiel) or HSA(L/mmol * s) (L/mmol * s) 36  1 3.7 22.1 25.3 37  2 6.1 29.8 35.7 38  32.9 38.2 51.5 39  4 3.5 27.1 29.7 40  5 4.2 20.0 22.4 41  6 6.5 23.225.8 42  7 5.0 31.1 37.4 43 16 0.71 38.0 38.3 44 17 0.55 40.6 41.4 97 302.1 35.3 39.1 98 31 1.8 32.2 36.5 99 32 2.7 36.5 40.1 100  33 1.2 40.044.6 101  34 1.6 31.8 34.8 102  35 2.5 40.1 45.7 103  30 1.9 34.6 39.9104  31 2.5 31.7 36.1 105  32 2.2 35.4 40.3 106  33 1.5 41.6 46.7 107 34 1.7 33.6 37.0 108  35 2.4 39.1 44.9 Vergleichssubstanz 1 Gd-DTPA 3613.39 13.97 Vergleichssubstanz 2 Gd-GlyMeDOTA 4.7 18.3 20.8[Key:]Beispiel = ExampleGd-Komplex (aus Beispiel) = Gd complex (from Example)Anzahl Gd/BSA = Gd/BSA numberVergleichssubstanz = Comparison substance

This example shows that the conjugates that are produced with thecompounds according to the invention have, surprisingly enough, a higherrelaxivity than the comparison substances despite their low number ofgadolinium atoms per biomolecule. Compared to comparison substance 2, itwas possible to increase the relaxivity by the special liganding of themacrocyclic ring.

The entire disclosures of all applications, patents and publications,cited herein and of corresponding German application No. 10135356.1,filed Jul. 20, 2001 is incorporated by reference herein.

The preceding examples can be repeated with similar success bysubstituting the generically or specifically described reactants and/oroperating conditions of this invention for those used in the precedingexamples.

From the foregoing description, one skilled in the art can easilyascertain the essential characteristics of this invention and, withoutdeparting from the spirit and scope thereof, can make various changesand modifications of the invention to adapt it to various usages andconditions.

1. Compounds of formula I

in which Z represents a hydrogen atom or at least two Z's represent ametal ion equivalent, B¹, B², B³, B⁴ are independently selected from thegroup consisting of hydrogen atoms and C₁₋₄-alkyl radicals, R¹, R², R³are independently selected from the group consisting of hydrogen atomsand straight, branched or cyclic, saturated or unsaturated C₁₋₁₀-alkylor aryl radicals, which optionally are substituted with a carboxyl group—SO₃H or —PO₃H₂, and whereby the alkyl chains of the C₁₋₁₀-alkylradicals optionally contain an aryl group and/or 1-2 oxygen atoms,provided that at least one of the radicals B¹, B², B³, B⁴, R¹, R² and R³does not represent a hydrogen atom, A represents a straight or branched,saturated or unsaturated C₁₋₃₀-hydrocarbon chain that optionallycontains 1-5 oxygen atoms, 1-5 nitrogen atoms and/or 1-5 —NR′ radicals,in which R′ is defined as R¹, R² and R³ but can be selectedindependently, which optionally is substituted with 1-3 carboxyl groups,1-3 —SO₃H, 1-3 —PO₃H₂ and/or 1-3 halogen atoms, in which optionally 1-3carbon atoms are present as carbonyl groups, whereby the chain or aportion of the chain can be arranged concentrically, and which isconfigured in such a way that X is connected via at least 3 atoms to thenitrogen to which A is bonded, and X represents a group that canparticipate in a reaction with a biomolecule, as well as their salts,provided that a) If B¹, B², B³ and B⁴ are hydrogen atoms and R¹, R² andR³ are —CH₂CH₂CO₂H, A-X together are not —CH(CO₂H)CH₂CH₂CO₂H, b) If B¹,B², B³ and B⁴ are hydrogen atoms and R¹, R² and R³ are methyl or R¹, R²and R³ are ethyl radicals, which optionally are substituted with acarboxy group, A does not represent the radical —CH(R⁷)—CO—NR⁵U⁶—, inwhich R⁵ stands for a hydrogen atom, a methyl or an ethyl radical, whichoptionally is substituted with 1 carboxy group, R⁷ stands for astraight-chain, branched, saturated or unsaturated C₁-C₃₀-alkyl chain,which optionally is interrupted by 1-10 oxygen atoms, I phenylene group,or 1-phenylenoxy group, and/or optionally is substituted by 1-5 hydroxygroups, 1-3 carboxy groups or 1 phenyl group, and U⁶ stands for astraight-chain, branched, saturated or unsaturated C₁-C₂₀-alkylene groupthat contains 1-5 imino groups, 1-3 phenylene groups, 1-3 phenylenoxygroups, 1-3 phenylenimino groups, 1-5 amide groups, 1-2 hydrazidegroups, 1-5 carbonyl groups, 1-5 ethylenoxy groups, 1 urea group, 1thiourea group, 1-2 carboxyalkylimino groups, 1-2 ester groups, 1-10oxygen atoms, 1-5 sulfur atoms and/or 1-5 nitrogen atoms and/or isoptionally substituted by 1-5 hydroxy groups, 1-2 mercapto groups, 1-5oxo groups, 1-5 thioxo groups, 1-3 carboxy groups, 1-5 carboxyalkylgroups, 1-5 ester groups and/or 1-3 amino groups, whereby the optionallycontained phenylene groups can be substituted by 1-2 carboxy groups, 1-2sulfone groups or 1-2 hydroxy groups, and c) If B¹, B², B³ and B⁴ arehydrogen atoms and R¹, R² and R³ represent the same C₁₋₄-alkyl radical,A does not represent the radical

in which R⁶ is a hydrogen atom or a C₁₋₄-alkyl radical, D is a saturatedor unsaturated, straight-chain or branched C₁₋₄-alkylene group, whichoptionally can be interrupted or substituted with a carbonyl group, andD is bonded to X.
 2. Compounds according to claim 1, in which R¹, R² andR³ are independently selected from the group consisting of hydrogenatoms, straight-chain or branched C₁₋₁₀-alkyl radicals, cyclohexylradicals, —CH₂—COOH, —C(CH₃)₂—COOH, phenyl radicals or radicals offormula —(CH₂)_(m)—(O)_(n)-(phenylene)_(p)-Y, in which m is an integerfrom 1 to 5, n is 0 or 1, p is 0 or 1, and y represents a hydrogen atom,a methoxy radical, a carboxyl group, —SO₃H or —PO₃H₂.
 3. Compoundsaccording to claim 2, in which if B¹, B², B³ and B⁴ are hydrogen atoms,R¹, R² and R³ are independently selected from the group consisting ofhydrogen atoms, isopropyl radicals, isobutyl radicals, tert-butylradicals, straight-chain or branched C₅₋₁₀-alkyl radicals, cyclohexylradicals, —CH₂—COOH, —C(CH₃)₂—COOH, phenyl radicals or radicals offormula —(CH₂)_(m)—(O)_(n)-(phenylene)_(p)-Y, in which m is an integerfrom 1 to 5, n is 0 or 1, p is 0 or 1, and Y represents a hydrogen atom,a methoxy radical, a carboxyl group, —SO₃H or —PO₃H₂, provided that atleast one of the radicals R¹, R² and R³ does not represent a hydrogenatom.
 4. Compounds according to claim 3, in which if B¹, B², B³ and B⁴are hydrogen atoms, R¹, R² and R³ are independently selected from thegroup consisting of hydrogen atoms, isopropyl, cyclohexyl or phenylradicals, provided that at least one of the radicals R¹, R² and R³ doesnot represent a hydrogen atom.
 5. Compounds according to claim 1, inwhich A represents a radical A′-U, in which A′ is bonded to the nitrogenatom of the macrocyclic ring and U is bonded to X, and whereby A′represents a) A bond, b) —CH(CO₂H)—, c) a group of formula

in which Q represents a hydrogen atom, a C₁₋₁₀-alkyl radical, whichoptionally is substituted with a carboxyl group, or an aryl radical,which optionally is substituted with a carboxyl group, a C₁₋₁₅-alkoxygroup, an aryloxy group or a halogen atom, and R′ is defined as R¹, R²and R³ in claim 1, but can be selected independently, or d) a group offormula

in which o is 0 or 1, and the ring optionally is annellated with abenzene ring, whereby this benzene ring, if present, can be substitutedwith a methoxy or carboxyl group, —SO₃H or —PO₃H₂, whereby in the groupsunder c) and d), the positions that are marked

are bonded to the adjacent groups, and in which position α is bonded toa nitrogen atom of the macrocyclic ring and position β is bonded to U,and U represents a straight or branched, saturated or unsaturatedC₁₋₃₀-hydrocarbon chain that optionally contains 1-3 oxygen atoms, 1-3nitrogen atoms and/or 1-3 —NR″ radicals, in which R″ is defined as R¹,R² and R³ in claim 1 but can be selected independently, and in whichoptionally 1-3 carbon atoms are present as carbonyl groups, whereby thechain or a portion of the chain can be arranged concentrically, providedthat A′ and U together are configured in such a way that X is bonded viaat least 3 atoms with the nitrogen atom to which A′ is bonded. 6.Compounds according to claim 5, in which for A′, the group of formula

is selected from —C(CH₃)H—CO—NH—, —C(phenyl)H—CO—NH— and—C(p-dodecanoxyphenyl)H—CO—NH—.
 7. Compounds according to claim 5, inwhich for A′, the group of formula

is selected from:

whereby R⁴ is —OCH₃, —CO₂—H, —SO₃H or —PO₃H₂.
 8. Compounds according toclaim 5, in which U is selected from —CH₂—, —(CH₂)₅—, —(CH₂)₁₀—,-phenylene-O—CH₂—, -phenylene-O—(CH₂)₃—, -phenylene-O—(CH₂)₁₀—,—CH₂-phenylene-, -cyclohexylene-O—CH₂—, -phenylene-, —C(phenyl)H—,—CH₂-pyridylene-O—CH₂—, —CH₂-pyridylene- and —CH₂—CO—NH—CH₂—CH₂— 9.Compounds according to claim 1, in which X is selected from the groupthat consists of carboxyl, activated carboxyl, amino, isocyanate,isothiocyanate, hydrazine, semicarbazide, thiosemicarbazide,chloroacetamide, bromoacetamide, iodoacetamide, acylamino, mixedanhydrides, azide, hydroxide, sulfonyl chloride, carbodiimide andradicals of formulas

in which Hal is a halogen atom.
 10. Compounds according to claim 9, inwhich the activated carboxyl group is selected from

11.10-(4-Carboxy-1-methyl-2-oxo-3-azabutyl)-1,4,7-α,α′,α″-trimethyl-1,4,7-tris(carboxymethyl)-1,4,7,10-tetraazacyclododecane,10-(4-carboxy-1-methyl-2-oxo-3-azabutyl)1,4,7-α,α′,α″-tris(isopropyl)-1,4,7-tris(carboxymethyl)-1,4,7,10-tetraazacyclododecane,10-(4-carboxy-1-methyl-2-oxo-3-azabutyl)-1,4,7-α,α′,α″-tris(cyclohexyl)-1,4,7-tris(carboxymethyl)-1,4,7,10-tetraazacyclododecane,10-(4-(t-butoxycarbonyl-1-phenyl-2-oxo-3-azabutyl)-1,4,7-α,α′,α″-trimethyl-1,4,7-tris(carboxymethyl)-1,4,7,10-tetraazacyclododecane,10-[α-(4-(ethoxycarbonylmethoxy)phenyl)-methoxycarbonylmethyl]-1,4,7-α,α′,α″-trimethyl-1,4,7-tris(carboxymethyl)-1,4,7,10-tetraazacyclododecane,10-[α-(4-(ethoxycarbonylpropoxy)phenyl)-methoxycarbonylmethyl]-1,4,7-α,α′,α″-trimethyl-1,4,7-tris(carboxymethyl)-1,4,7,10-tetraazacyclododecane,10-[α-(4-ethoxycarbonyldecyloxy)phenyl)-methoxycarbonylmethyl]-1,4,7-α,α′,α″-trimethyl-1,4,7-tris(carboxymethyl)-1,4,7,10-tetraazacyclododecane,10-(p-carboxybenzyl)-1,4,7-α,α′,α″-trimethyl-1,4,7-tris(carboxymethyl)-1,4,7,10-tetraazacyclododecane,10-(p-carboxybenzyl)-1,4,7-α,α′,α″-tris(isopropyl)-1,4,7-tris(carboxymethyl)-1,4,7,10-tetraazacyclododecane,10-(p-carboxybenzyl)-1,4,7-α,α′,α″-tris(cyclohexyl)-1,4,7-tris(carboxymethyl)-1,4,7,10-tetraazacyclododecane,10-(p-carboxybenzyl)-1,4,7-α,α′,α″-triphenyl-1,4,7-tris(carboxymethyl)-1,4,7,10-tetraazacyclododecane,10-(4-(t-butoxycarbony-1-phenyl-2-oxo-3-azabutyl)-1,4,7-α,α′,α″-triphenyl-1,4,7-tris(carboxymethyl)-1,4,7,10-tetraazacyclododecane,10-(4-carboxy-2-oxo-3-azabutyl)-1,4,7-α,α′,α″-tris(isopropyl)-1,4,7-tris(carboxymethyl)-1,4,7,10-tetraazacyclododecane,10-(4-carboxy-2-oxo-3-azabutyl)-1,4,7-α,α′,α″-tris(cyclohexyl)-1,4,7-tris(carboxymethyl)-1,4,7,10-tetraazacyclododecane,10-(4-carboxy-1-methyl-2-oxo-3-azabutyl)-2,5,8,11-tetramethyl-1,4,7,10-tetraazacyclododecane-1,4,7-triaceticacid-tri-tert-butyl ester,10-[8-(N-maleimido)-1-methyl-2,5-dioxo-3,6-diazaoctyl]-1,4,7-α,α′,α″-tris-(isopropyl)-1,4,7-tris(carboxymethyl)-1,4,7,10-tetraazacyclododecaneand10-[8-(N-maleimido)-1-methyl-2,5-dioxo-3,6-diazaoctyl]-1,4,7-α,α′,α″-tris(cyclohexyl)1,4,7-tris(carboxymethyl)-1,4,7,10-tetraazacyclododecane,10-[1-(carboxymethyl)-2-oxo-pyrrolidin-3-yl]-1,4,7-α,α′,α″-trimethyl-1,4,7-tris(carboxymethyl)-1,4,7,10-tetraazacyclododecane,10-[1-(carboxymethyl)-2-oxo-pyrrolidin-3-yl]-1,4,7-α,α′,α″-tris(isopropyl)-1,4,7-tris(carboxymethyl)-1,4,7,10-tetraazacyclododecane,10-[1-(carboxymethyl)-2-oxo-pyrrolidin-3-yl]-1,4,7-α,α′,α″-tris(cyclohexyl)-1,4,7-tris(carboxymethyl)-1,4,7,10-tetraazacyclododecane,10-[1-(4-carboxyphenyl)-2-oxo-pyrrolidin-3-yl]-1,4,7-α,α′,α″-trimethyl-1,4,7-tris(carboxymethyl)-1,4,7,10-tetraazacyclododecane,10-[1-(4-carboxyphenyl)-2-oxo-pyrrolidin-3-yl]-1,4,7-α,α′,α″-tris(isopropyl)-1,4,7-tris(carboxymethyl)-1,4,7,10-tetraazacyclododecane,10-[1-(4-carboxyphenyl)-2-oxo-pyrrolidin-3-yl]-1,4,7-α,α′,α″-tris(cyclohexyl)-1,4,7-tris(carboxymethyl)-1,4,7,10-tetraazacyclododecane,10-[1-(carboxymethyl)-2-oxo-piperidin-3-yl]-1,4,7-α,α′,α″-trimethyl-1,4,7-tris(carboxymethyl)-1,4,7,10-tetraazacyclododecane,10-[1-(carboxymethyl)-2-oxo-piperidin-3-yl]-1,4,7-α,α′,α″-tris(isopropyl)-1,4,7-tris(carboxymethyl)-1,4,7,10-tetraazacyclododecane,10-[1-(carboxymethyl)-2-oxo-piperidin-3-yl]-1,4,7-α,α′,α″-tris(cyclohexyl)-1,4,7-tris(carboxymethyl)-1,4,7,10-tetraazacyclododecane,10-[1-(4-carboxyphenyl)-2-oxo-piperidin-3-yl]-1,4,7-α,α′,α″-trimethyl-1,4,7-tris(carboxymethyl)-1,4,7,10-tetraazacyclododecane,10-[1-(4-carboxyphenyl)-2-oxo-piperidin-3-yl]-1,4,7-α,α′,α″-tris(isopropyl)-1,4,7-tris(carboxymethyl)-1,4,7,10-tetraazacyclododecane,10-[1-(4-carboxyphenyl)-2-oxo-piperidin-3-yl]-1,4,7-α,α′,α″-tris(cyclohexyl)-1,4,7-tris(carboxymethyl)-1,4,7,10-tetraazacyclododecane,10-[4-(benzyloxycarbonyl)-1-methyl-2-oxo-3-azabutyl]-1,7-α,α′-dimethyl-1,7-bis(t-butoxycarbonylmethyl)-4-α-isopropyl-4-benzyloxycarbonylmethyl-1,4,7,10-tetraazacyclododecane,10-[4-(Benzyloxycarbonyl)-1-methyl-2-oxo-3-azabutyl]-1,7-α,α′-diisopropyl-1,7-bis(t-butoxycarbonylmethyl)-4-α-methyl-4-benzyloxycarbonylmethyl-1,4,7,10-tetraazacyclododecane,10-[4-(benzyloxycarbonyl)-1-methyl-2-oxo-3-azabutyl]-1,7-α,α′-dimethyl-1,7-bis(t-butoxycarbonylmethyl)-4-α-cyclohexyl-4-benzyloxycarbonylmethyl-1,4,7,10-tetraazacyclododecane,10-[α-(4-(ethoxycarbonylmethoxy)phenyl)-methoxycarbonylmethyl]-1,7-α,α′-dimethyl-1,7-bis(t-butoxycarbonylmethyl)-4-α-isopropyl-4-benzyloxycarbonylmethyl-1,4,7,10-tetraazacyclododecane,10-[4-(benzyloxycarbonyl)-2-oxo-3-azabutyl]-1,7-α-α′-diisopropyl-1,7-bis(t-butoxycarbonylmethyl)-4-α-methyl-4-benzyloxycarbonylmethyl-1,4,7,10-tetraazacyclododecane,10-[4-(benzyloxycarbonyl)-2-oxo-3-azabutyl]-1,7-α-α′-dimethyl-1,7-bis(t-butoxycarbonylmethyl)-4-α-cyclohexyl-4-benzyloxycarbonylmethyl-1,4,7,10-tetraazacyclododecane.12. Compounds according to claim 1, in which at least two of radicals Zstand for a metal ion equivalent of a radioactive or paramagneticelement of atomic numbers 21-29, 31, 32, 37-39, 42-44, 46, 47, 49,58-71, 75, 77, 82 or
 83. 13. Use of the compounds of formula I

in which Z, B¹, B², B³, B⁴, R¹, R², R³, A and X are defined as in claim1, provided that at least one of the B¹, B², B³, B⁴, R¹, R² and R³ doesnot represent a hydrogen atom and if B¹, B², B³, B⁴ are hydrogen atomsand R¹, R², R³ represent the same C₁₋₄-alkyl radicals, A does notrepresent the radical

in which R⁶ is a hydrogen atom or a C₁₋₄-alkyl radical, D is a saturatedor unsaturated, straight-chain or branched C₁₋₄-alkylene group, whichoptionally is interrupted or substituted with a carbonyl group, and D isbonded to X, for the production of a conjugate with a biomolecule. 14.Use according to claim 13, in which the biomolecule is selected from thegroup that consists of biopolymers, proteins, synthetically modifiedbiopolymers, carbohydrates, antibodies, DNA and RNA fragments, β-aminoacids, vector amines for transfer into the cell, biogenic amines,pharmaceutical agents, oncological preparations, synthetic polymers,which are directed to a biological target, steroids, prostaglandins,taxol and derivatives thereof, endothelins, alkaloids, folic acid andderivatives thereof, bioactive lipids, fats, fatty acid esters,synthetically modified mono-, di- and triglycerides, liposomes, whichare derivatized on the surface, micelles that consist of natural fattyacids or perfluoroalkyl compounds, porphyrins, texaphrines, expandedporphyrins, cytochromes, inhibitors, neuramidases, neuropeptides,immunomodulators, endoglycosidases, substrates that are attacked by theenzymes calmodulin kinase, casein-kinase II, glutathione-S-transferase,heparinase, matrix-metalloproteases, β-insulin-receptor-kinase,UDP-galactose 4-epimerase, fucosidases, G-proteins, galactosidases,glycosidases, glycosyltransferases and xylosidase, antibiotics, vitaminsand vitamin analogs, hormones, DNA intercalators, nucleosides,nucleotides, lectins, vitamin B12, Lewis-X and related substances,psoralens, dienetriene antibiotics, carbacyclins, VEGF, somatostatin andderivatives thereof, biotin derivatives, antihormones, tumor-specificproteins and synthetic agents, polymers that accumulate in acidic orbasic areas of the body, myoglobins, apomyoglobins, neurotransmitterpeptides, tumor necrosis factors, peptides that accumulate in inflamedtissues, blood-pool reagents, anion and cation-transporter proteins,polyesters, polyamides and polyphosphates.
 15. Process for theproduction of compounds of formula I

in which Z, B¹, B², B³, B⁴, R¹, R², R³, A and X are defined as in claim1, provided that at least one of the B¹, B², B³, B⁴, R¹, R² and R³ doesnot represent a hydrogen atom and if B¹, B², B³, B⁴ are hydrogen atomsand R¹, R², R³ represent the same C₁₋₄-alkyl radicals, A does notrepresent the radical

in which R⁶ is a hydrogen atom or a C₁₋₄-alkyl radical, D is a saturatedor unsaturated, straight-chain or branched C₁₋₄-alkylene group, whichoptionally is interrupted or substituted with a carbonyl group, and D isbonded to X, in which a compound of formula II

in which B¹, B², B³ and B⁴ are defined as in claim 1, is optionallyreacted with Nu-A-X′, Nu-CH(R¹)—CO₂-Z′, Nu-CH(R²)—CO₂-Z′ andNu-CH(R³)—CO₂-Z′ optionally after protective groups for the nitrogenatoms are introduced, whereby A and R¹, R² and R³ are defined as inclaim 1, and Nu is a nucleofuge, X′ stands for X or a protected form ofX, and X is defined as in claim 1, and Z′ stands for a hydrogen atom, ametal ion equivalent or a protective group for carboxyl, then theoptionally present protective groups are removed, and, if desired,reacted in a way that is known in the art with at least one metal oxideor metal salt of a desired element and optionally then still presentacid hydrogen atoms are completely or partially substituted in the thusobtained complexes by cations of inorgnaic and/or organic bases, aminoacids or amino acid amides.